328356 SRR1182054 SRP039090 SRS565417 SRX480479 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 18 Weeks Old Fetus (F120) Frontal Lobe - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;18 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;18 Weeks Old Fetus (F120) Frontal Lobe|sex;;male|tissue;;Frontal Lobe 200 18 Weeks Old Fetus (F120) Frontal Lobe 1868607200 9343036 2017-01-20 00:00:00 1205929983 1868607200 9343036 2 9343036 index:0,count:9343036,average:100,stdev:0|index:1,count:9343036,average:100,stdev:0 18 Weeks Old Fetus (F120) Frontal Lobe - RNA-seq Stanford University 0.34 2.45 0.04 1814177375 2446912303 1703241825 2332833584 134.88 136.96 9156178 7946183 325.352 1431.649 261 64492 73.48 78.26 11140425 6728231 11140425 6728231 71.45 73.19 11140425 6542130 11140425 6291802 217470999 11.99 1.49 0 5.98 0 0.14 0 0.05 0 0.00 0 1.82 0 9156178 0 200 0 198.31 0 1.89 0 0.01 0 1.65 0 0.01 0 300.31 0 0.35 0 138933 0 9343036 0 559122 0 12752 0 4292 0 0 0 169814 0 1762 0 0 0 21788 0 3295227 0 12383 0 3331160 0 92.02 0 8597056 0 150324 3510103 23.350250126394 9343036.0 9156178.0 138933.0 559122.0 12752.0 4292.0 0.0 169814.0 8597056.0 98.0 1.5 6.0 0.1 0.0 0.0 1.8 92.0 100 100 100.00 38 934303600 21.1 27.9 27.6 23.5 0.0 35.8 21.1 bulk 328388 SRR1182058 SRP039090 SRS565419 SRX480483 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 18 Weeks Old Fetus (F120) Liver - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;18 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;18 Weeks Old Fetus (F120) Liver|sex;;male|tissue;;Liver 200 18 Weeks Old Fetus (F120) Liver 2720343400 13601717 2017-01-20 00:00:00 1734656197 2720343400 13601717 2 13601717 index:0,count:13601717,average:100,stdev:0|index:1,count:13601717,average:100,stdev:0 18 Weeks Old Fetus (F120) Liver - RNA-seq Stanford University 0.52 0.77 0.01 2674363640 3511070584 1886452581 2705514586 131.29 143.42 13482081 11611886 323.563 979.605 247 106630 67.95 96.23 28001313 9160775 28001313 9160775 81.08 89.88 28001313 10931540 28001313 8555958 35647096 1.33 0.94 0 29.13 0 0.04 0 0.01 0 0.00 0 0.83 0 13482081 0 200 0 198.32 0 1.87 0 0.01 0 1.76 0 0.00 0 218.60 0 0.28 0 127648 0 13601717 0 3962460 0 5878 0 950 0 0 0 112808 0 2371 0 0 0 31879 0 5760298 0 16592 0 5811140 0 69.99 0 9519621 0 152715 9252195 60.584716629015 13601717.0 13482081.0 127648.0 3962460.0 5878.0 950.0 0.0 112808.0 9519621.0 99.1 0.9 29.1 0.0 0.0 0.0 0.8 70.0 100 100 100.00 38 1360171700 19.9 28.3 29.4 22.3 0.0 35.8 22.2 bulk 328452 SRR1182060 SRP039090 SRS565420 SRX480485 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 18 Weeks Old Fetus (F120) Lung - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;18 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;18 Weeks Old Fetus (F120) Lung|sex;;male|tissue;;Lung 200 18 Weeks Old Fetus (F120) Lung 2444433600 12222168 2017-01-20 00:00:00 1559122702 2444433600 12222168 2 12222168 index:0,count:12222168,average:100,stdev:0|index:1,count:12222168,average:100,stdev:0 18 Weeks Old Fetus (F120) Lung - RNA-seq Stanford University 0.36 1.78 0.02 2396315921 3255103075 2121989528 3016919273 135.84 142.17 12087452 10013382 327.002 1440.536 238 86924 83.52 94.26 18683394 10095538 18683394 10095538 82.73 88.85 18683394 9999420 18683394 9515384 57598015 2.40 1.09 0 11.27 0 0.06 0 0.02 0 0.00 0 1.03 0 12087452 0 200 0 198.29 0 1.98 0 0.01 0 1.76 0 0.00 0 312.06 0 0.29 0 133150 0 12222168 0 1377639 0 6913 0 1866 0 0 0 125937 0 2977 0 0 0 35045 0 6707117 0 13650 0 6758789 0 87.63 0 10709813 0 181333 7111077 39.215570249210 12222168.0 12087452.0 133150.0 1377639.0 6913.0 1866.0 0.0 125937.0 10709813.0 98.9 1.1 11.3 0.1 0.0 0.0 1.0 87.6 100 100 100.00 38 1222216800 20.3 28.7 28.2 22.8 0.0 35.9 22.0 bulk 328468 SRR1182062 SRP039090 SRS565421 SRX480487 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 18 Weeks Old Fetus (F120) Skeletal Muscle - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;18 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;18 Weeks Old Fetus (F120) Skeletal Muscle|sex;;male|tissue;;Skeletal Muscle 200 18 Weeks Old Fetus (F120) Skeletal Muscle 3313323600 16566618 2017-01-20 00:00:00 2094100382 3313323600 16566618 2 16566618 index:0,count:16566618,average:100,stdev:0|index:1,count:16566618,average:100,stdev:0 18 Weeks Old Fetus (F120) Skeletal Muscle - RNA-seq Stanford University 0.56 1.8 0.02 3245660855 4531300677 2969203993 4220045243 139.61 142.13 16393624 13917817 317.311 1164.761 232 137751 86.47 94.49 21074584 14176089 21074584 14176089 86.56 89.51 21074584 14190297 21074584 13429144 76796254 2.37 1.08 0 8.39 0 0.04 0 0.01 0 0.00 0 0.99 0 16393624 0 200 0 198.10 0 1.83 0 0.01 0 1.81 0 0.00 0 315.55 0 0.28 0 179164 0 16566618 0 1390525 0 6861 0 1820 0 0 0 164313 0 2976 0 0 0 53422 0 10697032 0 21906 0 10775336 0 90.56 0 15003099 0 183093 11341005 61.941226589766 16566618.0 16393624.0 179164.0 1390525.0 6861.0 1820.0 0.0 164313.0 15003099.0 99.0 1.1 8.4 0.0 0.0 0.0 1.0 90.6 100 100 100.00 38 1656661800 20.1 28.5 28.0 23.4 0.0 36.0 22.2 bulk 328484 SRR1182064 SRP039090 SRS565422 SRX480489 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 18 Weeks Old Fetus (F120) Small Intestine - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;18 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;18 Weeks Old Fetus (F120) Small Intestine|sex;;male|tissue;;Small Intestine 200 18 Weeks Old Fetus (F120) Small Intestine 2365967600 11829838 2017-01-20 00:00:00 1505988086 2365967600 11829838 2 11829838 index:0,count:11829838,average:100,stdev:0|index:1,count:11829838,average:100,stdev:0 18 Weeks Old Fetus (F120) Small Intestine - RNA-seq Stanford University 0.52 1.58 0.03 2314107999 3224894891 2080486319 3013955453 139.36 144.87 11676328 10087523 316.120 1118.391 238 92829 81.24 90.32 16951383 9486127 16951383 9486127 79.51 84.38 16951383 9284404 16951383 8862320 112269359 4.85 1.00 0 9.92 0 0.06 0 0.02 0 0.00 0 1.22 0 11676328 0 200 0 198.26 0 1.97 0 0.01 0 1.74 0 0.00 0 325.09 0 0.33 0 118062 0 11829838 0 1174081 0 6620 0 2261 0 0 0 144629 0 2453 0 0 0 30836 0 5876222 0 12487 0 5921998 0 88.78 0 10502247 0 161591 6227967 38.541546249482 11829838.0 11676328.0 118062.0 1174081.0 6620.0 2261.0 0.0 144629.0 10502247.0 98.7 1.0 9.9 0.1 0.0 0.0 1.2 88.8 100 100 100.00 38 1182983800 20.3 28.4 28.1 23.2 0.0 35.9 21.5 bulk 328509 SRR1182067 SRP039090 SRS565423 SRX480493 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 38 Weeks Old Fetus (F122) Frontal Lobe - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;38 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;38 Weeks Old Fetus (F122) Frontal Lobe|sex;;male|tissue;;Frontal Lobe 200 38 Weeks Old Fetus (F122) Frontal Lobe 3067114600 15335573 2017-01-20 00:00:00 1957459625 3067114600 15335573 2 15335573 index:0,count:15335573,average:100,stdev:0|index:1,count:15335573,average:100,stdev:0 38 Weeks Old Fetus (F122) Frontal Lobe - RNA-seq Stanford University 0.85 2.15 0.03 3010529296 4140895535 2742922995 3896552450 137.55 142.06 15174119 12563969 322.042 1879.143 253 121590 85.24 93.53 21420423 12934999 21420423 12934999 83.1 87.76 21420423 12610080 21420423 12137802 75645568 2.51 1.05 0 8.76 0 0.05 0 0.01 0 0.00 0 0.99 0 15174119 0 200 0 198.47 0 1.91 0 0.01 0 1.74 0 0.00 0 322.85 0 0.29 0 160274 0 15335573 0 1344103 0 7527 0 2090 0 0 0 151837 0 3611 0 0 0 46049 0 6920729 0 18094 0 6988483 0 90.18 0 13830016 0 194867 7275323 37.334812975003 15335573.0 15174119.0 160274.0 1344103.0 7527.0 2090.0 0.0 151837.0 13830016.0 98.9 1.0 8.8 0.0 0.0 0.0 1.0 90.2 100 100 100.00 38 1533557300 21.1 27.9 27.7 23.3 0.0 36.0 22.1 bulk 328583 SRR1182070 SRP039090 SRS565424 SRX480496 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 38 Weeks Old Fetus (F122) Liver - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;38 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;38 Weeks Old Fetus (F122) Liver|sex;;male|tissue;;Liver 200 38 Weeks Old Fetus (F122) Liver 3634541000 18172705 2017-01-20 00:00:00 2318674147 3634541000 18172705 2 18172705 index:0,count:18172705,average:100,stdev:0|index:1,count:18172705,average:100,stdev:0 38 Weeks Old Fetus (F122) Liver - RNA-seq Stanford University 0.48 0.7 0.01 3568890383 4835470731 2915127863 4119507863 135.49 141.31 18006921 15175289 327.845 972.150 239 144105 79.91 97.76 29680934 14388932 29680934 14388932 83.4 89.45 29680934 15017423 29680934 13165682 31908024 0.89 0.98 0 18.10 0 0.03 0 0.00 0 0.00 0 0.88 0 18006921 0 200 0 198.22 0 1.94 0 0.01 0 1.73 0 0.00 0 231.99 0 0.30 0 177682 0 18172705 0 3288657 0 5825 0 773 0 0 0 159186 0 2459 0 0 0 74256 0 9325199 0 22703 0 9424617 0 80.99 0 14718264 0 152060 11778891 77.462126792056 18172705.0 18006921.0 177682.0 3288657.0 5825.0 773.0 0.0 159186.0 14718264.0 99.1 1.0 18.1 0.0 0.0 0.0 0.9 81.0 100 100 100.00 38 1817270500 20.2 28.1 28.8 22.9 0.0 35.9 22.4 bulk 328599 SRR1182072 SRP039090 SRS565425 SRX480498 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 38 Weeks Old Fetus (F122) Lung - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;38 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;38 Weeks Old Fetus (F122) Lung|sex;;male|tissue;;Lung 200 38 Weeks Old Fetus (F122) Lung 3327413800 16637069 2017-01-20 00:00:00 2113785636 3327413800 16637069 2 16637069 index:0,count:16637069,average:100,stdev:0|index:1,count:16637069,average:100,stdev:0 38 Weeks Old Fetus (F122) Lung - RNA-seq Stanford University 1.11 2.2 0.03 3257681512 4528044108 3029213069 4312755781 139.0 142.37 16441426 13941298 315.657 1258.243 238 143664 85.56 92.0 20961612 14067822 20961612 14067822 83.58 86.64 20961612 13741314 20961612 13248128 119408149 3.67 0.99 0 6.91 0 0.05 0 0.05 0 0.00 0 1.08 0 16441426 0 200 0 198.25 0 1.99 0 0.01 0 1.83 0 0.00 0 302.49 0 0.31 0 164997 0 16637069 0 1149748 0 8747 0 7501 0 0 0 179395 0 3198 0 0 0 46304 0 9307640 0 20067 0 9377209 0 91.91 0 15291678 0 206019 9760150 47.374999393260 16637069.0 16441426.0 164997.0 1149748.0 8747.0 7501.0 0.0 179395.0 15291678.0 98.8 1.0 6.9 0.1 0.0 0.0 1.1 91.9 100 100 100.00 38 1663706900 20.7 28.1 28.0 23.1 0.0 36.0 22.0 bulk 328614 SRR1182074 SRP039090 SRS565426 SRX480500 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 38 Weeks Old Fetus (F122) Skeletal Muscle - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;38 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;38 Weeks Old Fetus (F122) Skeletal Muscle|sex;;male|tissue;;Skeletal Muscle 200 38 Weeks Old Fetus (F122) Skeletal Muscle 3602839400 18014197 2017-01-20 00:00:00 2281578254 3602839400 18014197 2 18014197 index:0,count:18014197,average:100,stdev:0|index:1,count:18014197,average:100,stdev:0 38 Weeks Old Fetus (F122) Skeletal Muscle - RNA-seq Stanford University 1.16 1.5 0.02 3533038602 4937425564 3161762100 4483952588 139.75 141.82 17833145 14528829 331.507 1266.170 247 150618 85.86 95.9 23840577 15311406 23840577 15311406 87.77 91.24 23840577 15651453 23840577 14567307 59466153 1.68 1.08 0 10.37 0 0.05 0 0.01 0 0.00 0 0.94 0 17833145 0 200 0 198.22 0 1.82 0 0.01 0 1.73 0 0.00 0 381.48 0 0.26 0 194052 0 18014197 0 1867505 0 9705 0 1279 0 0 0 170068 0 2870 0 0 0 90571 0 11106647 0 22024 0 11222112 0 88.63 0 15965640 0 183452 12178883 66.387300220221 18014197.0 17833145.0 194052.0 1867505.0 9705.0 1279.0 0.0 170068.0 15965640.0 99.0 1.1 10.4 0.1 0.0 0.0 0.9 88.6 100 100 100.00 38 1801419700 20.1 28.2 27.4 24.3 0.0 36.1 22.7 bulk 328629 SRR1182076 SRP039090 SRS565427 SRX480502 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 38 Weeks Old Fetus (F122) Small Intestine - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;38 weeks fetus|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;38 Weeks Old Fetus (F122) Small Intestine|sex;;male|tissue;;Small Intestine 200 38 Weeks Old Fetus (F122) Small Intestine 3185245400 15926227 2017-01-20 00:00:00 2018101705 3185245400 15926227 2 15926227 index:0,count:15926227,average:100,stdev:0|index:1,count:15926227,average:100,stdev:0 38 Weeks Old Fetus (F122) Small Intestine - RNA-seq Stanford University 1.91 2.15 0.04 3119544567 4431954515 2931201120 4240801434 142.07 144.68 15739626 12923274 323.500 1335.741 238 108510 87.26 92.85 18851964 13734345 18851964 13734345 85.06 87.01 18851964 13388018 18851964 12869204 106923070 3.43 0.90 0 5.95 0 0.03 0 0.01 0 0.00 0 1.13 0 15739626 0 200 0 198.31 0 2.06 0 0.01 0 1.75 0 0.00 0 10.25 0 0.31 0 142634 0 15926227 0 948297 0 5532 0 1448 0 0 0 179621 0 3554 0 0 0 47318 0 8677484 0 17037 0 8745393 0 92.87 0 14791329 0 201936 9126179 45.193422668568 15926227.0 15739626.0 142634.0 948297.0 5532.0 1448.0 0.0 179621.0 14791329.0 98.8 0.9 6.0 0.0 0.0 0.0 1.1 92.9 100 100 100.00 38 1592622700 21.5 27.2 26.8 24.6 0.0 36.2 22.2 bulk 328652 SRR1182079 SRP039090 SRS565428 SRX480505 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Cerebellum - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Cerebellum|sex;;male|tissue;;Cerebellum 200 25 Years Old Adult 1 (N37) Cerebellum 2043314800 10216574 2017-01-20 00:00:00 1280424381 2043314800 10216574 2 10216574 index:0,count:10216574,average:100,stdev:0|index:1,count:10216574,average:100,stdev:0 25 Years Old Adult 1 (N37) Cerebellum - RNA-seq Stanford University 0.06 1.36 0.01 1973419031 2692861969 1742064552 2505148096 136.46 143.8 9955803 8751630 298.266 1498.656 242 82533 79.07 89.52 16420152 7872123 16420152 7872123 75.46 83.0 16420152 7513124 16420152 7298685 96773510 4.90 1.05 0 11.38 0 0.12 0 0.03 0 0.00 0 2.41 0 9955803 0 200 0 198.33 0 1.97 0 0.01 0 1.73 0 0.00 0 311.69 0 0.35 0 107533 0 10216574 0 1162347 0 11977 0 2968 0 0 0 245826 0 2411 0 0 0 23815 0 4094022 0 11522 0 4131770 0 86.07 0 8793456 0 131375 4257136 32.404460513796 10216574.0 9955803.0 107533.0 1162347.0 11977.0 2968.0 0.0 245826.0 8793456.0 97.4 1.1 11.4 0.1 0.0 0.0 2.4 86.1 100 100 100.00 38 1021657400 19.1 29.9 29.6 21.3 0.0 35.8 21.5 bulk 328740 SRR1182084 SRP039090 SRS565429 SRX480510 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Colon - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Colon|sex;;male|tissue;;Colon 200 25 Years Old Adult 1 (N37) Colon 2272204800 11361024 2017-01-20 00:00:00 1424952913 2272204800 11361024 2 11361024 index:0,count:11361024,average:100,stdev:0|index:1,count:11361024,average:100,stdev:0 25 Years Old Adult 1 (N37) Colon - RNA-seq Stanford University 0.03 1.68 0.01 2200157820 3061014765 1960192329 2845616828 139.13 145.17 11119901 9280648 323.424 3111.152 254 85474 87.27 97.92 17110623 9704889 17110623 9704889 84.58 91.15 17110623 9404755 17110623 9033425 18877625 0.86 1.17 0 10.64 0 0.05 0 0.05 0 0.00 0 2.02 0 11119901 0 200 0 197.94 0 1.92 0 0.01 0 1.99 0 0.00 0 307.52 0 0.32 0 133394 0 11361024 0 1209000 0 5837 0 5596 0 0 0 229690 0 2857 0 0 0 25156 0 6004880 0 13001 0 6045894 0 87.24 0 9910901 0 115888 6280728 54.196534585117 11361024.0 11119901.0 133394.0 1209000.0 5837.0 5596.0 0.0 229690.0 9910901.0 97.9 1.2 10.6 0.1 0.0 0.0 2.0 87.2 100 100 100.00 38 1136102400 18.4 30.3 30.4 20.9 0.0 35.8 21.9 bulk 328764 SRR1182087 SRP039090 SRS565430 SRX480513 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Frontal Lobe - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Frontal Lobe|sex;;male|tissue;;Frontal Lobe 200 25 Years Old Adult 1 (N37) Frontal Lobe 2025400200 10127001 2017-01-20 00:00:00 1285165573 2025400200 10127001 2 10127001 index:0,count:10127001,average:100,stdev:0|index:1,count:10127001,average:100,stdev:0 25 Years Old Adult 1 (N37) Frontal Lobe - RNA-seq Stanford University 0.11 1.55 0.01 1980917638 2754684306 1780629532 2584820786 139.06 145.16 9983626 8340156 323.652 1793.448 280 70105 85.6 95.19 15560967 8546150 15560967 8546150 82.22 89.19 15560967 8208787 15560967 8008013 39852987 2.01 1.10 0 9.93 0 0.08 0 0.02 0 0.00 0 1.32 0 9983626 0 200 0 198.48 0 1.87 0 0.01 0 1.74 0 0.00 0 4.93 0 0.28 0 111105 0 10127001 0 1005505 0 8114 0 1991 0 0 0 133270 0 2584 0 0 0 27931 0 4592899 0 11116 0 4634530 0 88.66 0 8978121 0 130157 4724355 36.297356269736 10127001.0 9983626.0 111105.0 1005505.0 8114.0 1991.0 0.0 133270.0 8978121.0 98.6 1.1 9.9 0.1 0.0 0.0 1.3 88.7 100 100 100.00 38 1012700100 19.1 29.9 30.2 20.8 0.0 35.7 21.7 bulk 328844 SRR1182091 SRP039090 SRS565431 SRX480516 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Heart - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Heart|sex;;male|tissue;;Heart 200 25 Years Old Adult 1 (N37) Heart 1948120800 9740604 2017-01-20 00:00:00 1224038116 1948120800 9740604 2 9740604 index:0,count:9740604,average:100,stdev:0|index:1,count:9740604,average:100,stdev:0 25 Years Old Adult 1 (N37) Heart - RNA-seq Stanford University 0.05 0.59 0.0 1905453165 2499582085 1475706265 2166536100 131.18 146.81 9600134 8002082 327.617 1228.508 242 65909 75.63 97.51 22093202 7260730 22093202 7260730 73.47 90.16 22093202 7052913 22093202 6713388 19046067 1.00 1.03 0 22.12 0 0.06 0 0.01 0 0.00 0 1.37 0 9600134 0 200 0 198.33 0 1.91 0 0.01 0 1.76 0 0.00 0 333.96 0 0.27 0 100095 0 9740604 0 2154362 0 6328 0 597 0 0 0 133545 0 1624 0 0 0 35854 0 4512682 0 9295 0 4559455 0 76.44 0 7445772 0 102213 4713800 46.117421463023 9740604.0 9600134.0 100095.0 2154362.0 6328.0 597.0 0.0 133545.0 7445772.0 98.6 1.0 22.1 0.1 0.0 0.0 1.4 76.4 100 100 100.00 38 974060400 18.5 30.8 29.1 21.6 0.0 35.9 22.2 bulk 328868 SRR1182094 SRP039090 SRS565432 SRX480518 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Liver - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Liver|sex;;male|tissue;;Liver 200 25 Years Old Adult 1 (N37) Liver 3427276000 17136380 2017-01-20 00:00:00 2130494244 3427276000 17136380 2 17136380 index:0,count:17136380,average:100,stdev:0|index:1,count:17136380,average:100,stdev:0 25 Years Old Adult 1 (N37) Liver - RNA-seq Stanford University 1.3 0.61 0.01 3356119056 4489210090 2757564658 3998824709 133.76 145.01 16943052 15095722 286.571 830.247 208 156435 76.14 92.59 33213250 12899879 33213250 12899879 74.19 85.45 33213250 12570356 33213250 11905846 108645651 3.24 0.81 0 17.57 0 0.07 0 0.01 0 0.00 0 1.04 0 16943052 0 200 0 198.34 0 1.85 0 0.01 0 1.74 0 0.00 0 10.11 0 0.27 0 138375 0 17136380 0 3010070 0 12291 0 2077 0 0 0 178960 0 2668 0 0 0 39715 0 7753726 0 16440 0 7812549 0 81.31 0 13932982 0 151008 8372261 55.442499735113 17136380.0 16943052.0 138375.0 3010070.0 12291.0 2077.0 0.0 178960.0 13932982.0 98.9 0.8 17.6 0.1 0.0 0.0 1.0 81.3 100 100 100.00 38 1713638000 20.0 28.7 28.5 22.8 0.0 36.1 22.5 bulk 328909 SRR1182099 SRP039090 SRS565433 SRX480523 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Lung - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Lung|sex;;male|tissue;;Lung 200 25 Years Old Adult 1 (N37) Lung 3419147200 17095736 2017-01-20 00:00:00 2117679374 3419147200 17095736 2 17095736 index:0,count:17095736,average:100,stdev:0|index:1,count:17095736,average:100,stdev:0 25 Years Old Adult 1 (N37) Lung - RNA-seq Stanford University 0.68 1.71 0.02 3335975348 4553155943 3072610159 4319969084 136.49 140.6 16861021 15097677 287.450 1180.138 214 154150 83.06 90.15 23108763 14003974 23108763 14003974 80.62 84.59 23108763 13593178 23108763 13139394 164287997 4.92 1.04 0 7.77 0 0.07 0 0.02 0 0.00 0 1.28 0 16861021 0 200 0 198.27 0 1.91 0 0.01 0 1.72 0 0.00 0 222.18 0 0.29 0 178092 0 17095736 0 1327664 0 12808 0 3457 0 0 0 218450 0 3024 0 0 0 41140 0 8807708 0 32389 0 8884261 0 90.86 0 15533357 0 197701 9188941 46.478980885276 17095736.0 16861021.0 178092.0 1327664.0 12808.0 3457.0 0.0 218450.0 15533357.0 98.6 1.0 7.8 0.1 0.0 0.0 1.3 90.9 100 100 100.00 38 1709573600 20.4 28.4 28.1 23.1 0.0 36.2 22.4 bulk 329767 SRR1182104 SRP039090 SRS565434 SRX480528 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Pancreas - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Pancreas|sex;;male|tissue;;Pancreas 200 25 Years Old Adult 1 (N37) Pancreas 2976845000 14884225 2017-01-20 00:00:00 1865303023 2976845000 14884225 2 14884225 index:0,count:14884225,average:100,stdev:0|index:1,count:14884225,average:100,stdev:0 25 Years Old Adult 1 (N37) Pancreas - RNA-seq Stanford University 0.18 0.35 0.01 2926905789 4128642375 2393510780 3482723962 141.06 145.51 14766654 11822903 345.557 1192.122 226 136169 80.14 97.96 23638458 11833720 23638458 11833720 87.21 93.39 23638458 12878420 23638458 11280886 27253670 0.93 0.83 0 18.05 0 0.03 0 0.00 0 0.00 0 0.76 0 14766654 0 200 0 198.24 0 1.84 0 0.00 0 1.79 0 0.00 0 318.95 0 0.26 0 122910 0 14884225 0 2686997 0 3788 0 617 0 0 0 113166 0 570 0 0 0 15009 0 10852542 0 12930 0 10881051 0 81.16 0 12079657 0 96499 14535196 150.625353630608 14884225.0 14766654.0 122910.0 2686997.0 3788.0 617.0 0.0 113166.0 12079657.0 99.2 0.8 18.1 0.0 0.0 0.0 0.8 81.2 100 100 100.00 38 1488422500 20.0 27.8 30.4 21.8 0.0 35.9 21.6 bulk 329790 SRR1182107 SRP039090 SRS565435 SRX480531 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Skeletal Muscle - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Skeletal Muscle|sex;;male|tissue;;Skeletal Muscle 200 25 Years Old Adult 1 (N37) Skeletal Muscle 3710061800 18550309 2017-01-20 00:00:00 2288545471 3710061800 18550309 2 18550309 index:0,count:18550309,average:100,stdev:0|index:1,count:18550309,average:100,stdev:0 25 Years Old Adult 1 (N37) Skeletal Muscle - RNA-seq Stanford University 1.43 0.94 0.01 3639706087 5026586686 3284169906 4700264537 138.1 143.12 18390055 15865693 300.029 945.420 208 156236 87.81 97.27 27375584 16147616 27375584 16147616 86.13 92.52 27375584 15838587 27375584 15359252 42331324 1.16 1.04 0 9.64 0 0.04 0 0.01 0 0.00 0 0.82 0 18390055 0 200 0 198.21 0 1.84 0 0.00 0 1.69 0 0.00 0 362.94 0 0.23 0 192058 0 18550309 0 1788486 0 7095 0 951 0 0 0 152208 0 2668 0 0 0 114821 0 11970891 0 20587 0 12108967 0 89.49 0 16601569 0 153288 12550733 81.876813579667 18550309.0 18390055.0 192058.0 1788486.0 7095.0 951.0 0.0 152208.0 16601569.0 99.1 1.0 9.6 0.0 0.0 0.0 0.8 89.5 100 100 100.00 38 1855030900 19.2 28.9 28.2 23.7 0.0 36.3 23.3 bulk 329862 SRR1182110 SRP039090 SRS565437 SRX480535 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Small Intestine - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Small Intestine|sex;;male|tissue;;Small Intestine 200 25 Years Old Adult 1 (N37) Small Intestine 1668923800 8344619 2017-01-20 00:00:00 1045057677 1668923800 8344619 2 8344619 index:0,count:8344619,average:100,stdev:0|index:1,count:8344619,average:100,stdev:0 25 Years Old Adult 1 (N37) Small Intestine - RNA-seq Stanford University 0.04 1.31 0.01 1622652269 2198262316 1388030266 2003891336 135.47 144.37 8196046 7013306 314.704 3129.795 254 69672 82.67 96.58 14432884 6775585 14432884 6775585 79.88 89.52 14432884 6547153 14432884 6280685 24528573 1.51 0.99 0 14.14 0 0.07 0 0.03 0 0.00 0 1.68 0 8196046 0 200 0 198.00 0 2.02 0 0.01 0 1.95 0 0.00 0 326.53 0 0.34 0 82740 0 8344619 0 1180193 0 5668 0 2533 0 0 0 140372 0 1879 0 0 0 16591 0 3981857 0 9358 0 4009685 0 84.08 0 7015853 0 102532 4188007 40.845853001990 8344619.0 8196046.0 82740.0 1180193.0 5668.0 2533.0 0.0 140372.0 7015853.0 98.2 1.0 14.1 0.1 0.0 0.0 1.7 84.1 100 100 100.00 38 834461900 18.3 30.6 30.4 20.8 0.0 35.7 21.7 bulk 329895 SRR1182114 SRP039090 SRS565438 SRX480539 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. 25 Years Old Adult 1 (N37) Stomach - RNA-seq "The Illumina mRNA-seq library preparation workflow was followed with some modifications, as described previously by Tan et al. (2013). The library amplification step was performed with SYBR Green I on a real-time PCR machine to prevent over-amplification. The standard 6bp Illumina barcodes were added to each library in the final PCR step. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq 2000 (Illumina)." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;25 Years Old Adult 1 (N37) Stomach|sex;;male|tissue;;Stomach 200 25 Years Old Adult 1 (N37) Stomach 3120389800 15601949 2017-01-20 00:00:00 1960410099 3120389800 15601949 2 15601949 index:0,count:15601949,average:100,stdev:0|index:1,count:15601949,average:100,stdev:0 25 Years Old Adult 1 (N37) Stomach - RNA-seq Stanford University 0.31 0.31 0.0 2902301902 3253065557 1389221564 1831648519 112.09 131.85 14672061 11535072 310.326 1491.460 212 132191 45.72 95.44 46335038 6707828 46335038 6707828 81.94 90.88 46335038 12022463 46335038 6387116 33165181 1.14 0.71 0 48.99 0 4.86 0 0.01 0 0.00 0 1.09 0 14672061 0 200 0 198.05 0 1.88 0 0.01 0 1.79 0 0.00 0 248.53 0 0.27 0 110711 0 15601949 0 7643882 0 758452 0 1952 0 0 0 169484 0 1220 0 0 0 15542 0 4602130 0 10546 0 4629438 0 45.05 0 7028179 0 140346 20389671 145.281454405541 15601949.0 14672061.0 110711.0 7643882.0 758452.0 1952.0 0.0 169484.0 7028179.0 94.0 0.7 49.0 4.9 0.0 0.0 1.1 45.0 100 100 100.00 38 1560194900 20.7 27.0 30.7 21.5 0.0 36.1 23.0 bulk 329934 SRR1182119 SRP039090 SRS565439 SRX480544 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Adrenal Gland - RNA-seq "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Adrenal Gland|sex;;male|tissue;;Adrenal Gland 194 Young Adult 2 (N6) Adrenal Gland 2703878492 13937518 2017-01-20 00:00:00 1973496375 2703878492 13937518 2 13937518 index:0,count:13937518,average:97,stdev:0|index:1,count:13937518,average:97,stdev:0 Young Adult 2 (N6) Adrenal Gland - RNA-seq Stanford University 7.04 2.03 0.06 2342436844 3293126361 2176607707 3114559180 140.59 143.09 12451610 10750703 303.740 1222.370 221 84087 73.63 79.28 15360788 9168373 15360788 9168373 73.82 74.48 15360788 9192226 15360788 8612948 276406709 11.80 0.58 0 6.36 0 0.12 0 0.01 0 0.00 0 10.52 0 12451610 0 194 0 189.06 0 1.90 0 0.01 0 1.42 0 0.01 0 252.14 0 1.36 0 80164 0 13937518 0 887011 0 17349 0 1732 0 0 0 1466827 0 1879 0 0 0 23721 0 3697210 0 12047 0 3734857 0 82.97 0 11564599 0 150351 4342509 28.882475008480 13937518.0 12451610.0 80164.0 887011.0 17349.0 1732.0 0.0 1466827.0 11564599.0 89.3 0.6 6.4 0.1 0.0 0.0 10.5 83.0 97 97 97.00 38 1351939246 22.1 25.7 26.3 25.9 0.0 25.4 9.2 bulk 329989 SRR1182120 SRP039090 SRS565440 SRX480545 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Cerebellum - RNA-seq - (rep1) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Cerebellum|sex;;male|tissue;;Cerebellum 194 Young Adult 2 (N6) Cerebellum 1490431190 7682635 2017-01-20 00:00:00 936842042 1490431190 7682635 2 7682635 index:0,count:7682635,average:97,stdev:0|index:1,count:7682635,average:97,stdev:0 Young Adult 2 (N6) Cerebellum - RNA-seq - (rep1) Stanford University 5.91 2.56 0.08 1344850472 1757481241 1288044820 1706051422 130.68 132.45 7107525 6002073 363.117 1670.071 315 42525 65.76 68.68 8097380 4673708 8097380 4673708 64.27 65.36 8097380 4568309 8097380 4447621 238580089 17.74 2.76 0 3.94 0 0.13 0 0.05 0 0.00 0 7.30 0 7107525 0 194 0 189.95 0 1.70 0 0.01 0 1.57 0 0.01 0 314.29 0 1.03 0 212422 0 7682635 0 302760 0 9915 0 4190 0 0 0 561005 0 934 0 0 0 11993 0 1571391 0 7570 0 1591888 0 88.57 0 6804765 0 164012 1665981 10.157677487013 7682635.0 7107525.0 212422.0 302760.0 9915.0 4190.0 0.0 561005.0 6804765.0 92.5 2.8 3.9 0.1 0.1 0.0 7.3 88.6 97 97 97.00 38 745215595 25.4 25.0 24.2 25.4 0.0 32.1 13.4 bulk 329996 SRR1182121 SRP039090 SRS565440 SRX480546 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Cerebellum - RNA-seq - (rep2) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Cerebellum|sex;;male|tissue;;Cerebellum 194 Young Adult 2 (N6) Cerebellum 1254596836 6466994 2017-01-20 00:00:00 790438459 1254596836 6466994 2 6466994 index:0,count:6466994,average:97,stdev:0|index:1,count:6466994,average:97,stdev:0 Young Adult 2 (N6) Cerebellum - RNA-seq - (rep2) Stanford University 5.97 2.56 0.08 1132328379 1480928242 1083939724 1437090453 130.79 132.58 5987791 5019614 373.131 1759.447 324 34912 65.84 68.8 6832737 3942230 6832737 3942230 64.38 65.48 6832737 3854911 6832737 3751808 200328425 17.69 2.71 0 3.99 0 0.13 0 0.05 0 0.00 0 7.22 0 5987791 0 194 0 189.91 0 1.70 0 0.01 0 1.55 0 0.01 0 298.48 0 1.04 0 175306 0 6466994 0 258039 0 8520 0 3546 0 0 0 467137 0 745 0 0 0 10100 0 1322543 0 6343 0 1339731 0 88.60 0 5729752 0 155329 1401224 9.021007023801 6466994.0 5987791.0 175306.0 258039.0 8520.0 3546.0 0.0 467137.0 5729752.0 92.6 2.7 4.0 0.1 0.1 0.0 7.2 88.6 97 97 97.00 38 627298418 25.3 24.9 24.4 25.3 0.0 32.2 13.5 bulk 330005 SRR1182122 SRP039090 SRS565440 SRX480547 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Cerebellum - RNA-seq - (rep3) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Cerebellum|sex;;male|tissue;;Cerebellum 194 Young Adult 2 (N6) Cerebellum 1692052480 8721920 2017-01-20 00:00:00 1067566981 1692052480 8721920 2 8721920 index:0,count:8721920,average:97,stdev:0|index:1,count:8721920,average:97,stdev:0 Young Adult 2 (N6) Cerebellum - RNA-seq - (rep3) Stanford University 6.17 2.55 0.08 1553617857 2022222628 1487551756 1959309661 130.16 131.71 8191533 7367937 293.800 1169.332 237 60971 63.19 66.02 9391399 5176371 9391399 5176371 61.69 62.73 9391399 5053460 9391399 4918468 296289595 19.07 2.13 0 4.02 0 0.11 0 0.05 0 0.00 0 5.91 0 8191533 0 194 0 190.42 0 1.72 0 0.01 0 1.51 0 0.01 0 310.88 0 0.90 0 185551 0 8721920 0 351044 0 9979 0 4747 0 0 0 515661 0 985 0 0 0 12495 0 1673396 0 8863 0 1695739 0 89.89 0 7840489 0 167988 1769947 10.536151391766 8721920.0 8191533.0 185551.0 351044.0 9979.0 4747.0 0.0 515661.0 7840489.0 93.9 2.1 4.0 0.1 0.1 0.0 5.9 89.9 97 97 97.00 38 846026240 26.0 24.3 23.5 26.2 0.0 33.0 14.3 bulk 330013 SRR1182123 SRP039090 SRS565440 SRX480548 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Cerebellum - RNA-seq - (rep4) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Cerebellum|sex;;male|tissue;;Cerebellum 194 Young Adult 2 (N6) Cerebellum 3370334840 17372860 2017-01-20 00:00:00 2504148819 3370334840 17372860 2 17372860 index:0,count:17372860,average:97,stdev:0|index:1,count:17372860,average:97,stdev:0 Young Adult 2 (N6) Cerebellum - RNA-seq - (rep4) Stanford University 2.17 2.0 0.03 2201925928 2872810523 2029067464 2716211009 130.47 133.86 15417437 14789776 157.443 603.650 130 224487 67.01 72.77 20749417 10331319 20749417 10331319 64.71 67.59 20749417 9976885 20749417 9596017 287734320 13.07 1.00 0 7.02 0 0.25 0 0.10 0 0.00 0 10.90 0 15417437 0 194 0 188.77 0 2.31 0 0.01 0 1.89 0 0.00 0 8.24 0 1.20 0 173472 0 17372860 0 1219956 0 43890 0 17400 0 0 0 1894133 0 2406 0 0 0 33701 0 4582433 0 27300 0 4645840 0 81.72 0 14197481 0 203798 4215346 20.683941942512 17372860.0 15417437.0 173472.0 1219956.0 43890.0 17400.0 0.0 1894133.0 14197481.0 88.7 1.0 7.0 0.3 0.1 0.0 10.9 81.7 97 97 97.00 38 1685167420 20.6 29.5 26.9 22.9 0.0 29.6 11.4 bulk 330021 SRR1182124 SRP039090 SRS565440 SRX480549 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Cerebellum - RNA-seq - (rep5) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Cerebellum|sex;;male|tissue;;Cerebellum 194 Young Adult 2 (N6) Cerebellum 2405650828 12400262 2017-01-20 00:00:00 1653717740 2405650828 12400262 2 12400262 index:0,count:12400262,average:97,stdev:0|index:1,count:12400262,average:97,stdev:0 Young Adult 2 (N6) Cerebellum - RNA-seq - (rep5) Stanford University 12.83 2.5 0.19 1468449587 1770826930 1411006449 1721049335 120.59 121.97 8757514 8416497 235.103 988.663 262 50063 37.03 38.73 10012452 3243260 10012452 3243260 37.06 37.5 10012452 3245254 10012452 3140235 564014328 38.41 8.09 0 3.09 0 0.15 0 0.05 0 0.00 0 29.17 0 8757514 0 194 0 185.38 0 1.66 0 0.01 0 1.84 0 0.01 0 217.76 0 1.06 0 1002772 0 12400262 0 382785 0 18933 0 6284 0 0 0 3617531 0 595 0 0 0 5101 0 595317 0 27726 0 628739 0 67.54 0 8374729 0 91955 594889 6.469349138165 12400262.0 8757514.0 1002772.0 382785.0 18933.0 6284.0 0.0 3617531.0 8374729.0 70.6 8.1 3.1 0.2 0.1 0.0 29.2 67.5 97 97 97.00 38 1202825414 30.1 20.7 22.8 26.2 0.1 28.2 10.8 bulk 330134 SRR1182132 SRP039090 SRS565443 SRX480557 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Frontal Lobe - RNA-seq "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Frontal Lobe|sex;;male|tissue;;Frontal Lobe 194 Young Adult 2 (N6) Frontal Lobe 3134172432 16155528 2017-01-20 00:00:00 2282459460 3134172432 16155528 2 16155528 index:0,count:16155528,average:97,stdev:0|index:1,count:16155528,average:97,stdev:0 Young Adult 2 (N6) Frontal Lobe - RNA-seq Stanford University 3.77 2.25 0.04 2854805232 3891580011 2660026384 3720255709 136.32 139.86 15127117 13111650 287.619 1310.860 221 117763 75.55 81.09 19891201 11428079 19891201 11428079 73.03 76.04 19891201 11046897 19891201 10716597 282941648 9.91 0.57 0 6.40 0 0.09 0 0.02 0 0.00 0 6.26 0 15127117 0 194 0 190.19 0 2.02 0 0.01 0 1.53 0 0.01 0 278.28 0 1.05 0 91464 0 16155528 0 1034176 0 13857 0 3445 0 0 0 1011109 0 2843 0 0 0 34514 0 5007550 0 14298 0 5059205 0 87.23 0 14092941 0 195433 5204820 26.632247368663 16155528.0 15127117.0 91464.0 1034176.0 13857.0 3445.0 0.0 1011109.0 14092941.0 93.6 0.6 6.4 0.1 0.0 0.0 6.3 87.2 97 97 97.00 38 1567086216 22.1 26.6 26.5 24.7 0.0 30.9 12.3 bulk 330172 SRR1182137 SRP039090 SRS565446 SRX480563 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Small Intestine - RNA-seq "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Small Intestine|sex;;male|tissue;;Small Intestine 194 Young Adult 2 (N6) Small Intestine 3641616098 18771217 2017-01-20 00:00:00 2323369138 3641616098 18771217 2 18771217 index:0,count:18771217,average:97,stdev:0|index:1,count:18771217,average:97,stdev:0 Young Adult 2 (N6) Small Intestine - RNA-seq Stanford University 4.88 1.82 0.05 3027425480 4148685353 2802091587 3933814226 137.04 140.39 16105504 13773304 291.411 2135.575 216 110291 77.75 84.04 20838339 12522785 20838339 12522785 76.52 78.98 20838339 12324504 20838339 11769545 256063200 8.46 0.45 0 6.41 0 0.08 0 0.02 0 0.00 0 14.10 0 16105504 0 194 0 190.04 0 2.12 0 0.01 0 1.45 0 0.01 0 181.17 0 1.04 0 85399 0 18771217 0 1203924 0 15085 0 4563 0 0 0 2646065 0 2555 0 0 0 33513 0 6007033 0 16543 0 6059644 0 79.39 0 14901580 0 200386 6324387 31.561022227102 18771217.0 16105504.0 85399.0 1203924.0 15085.0 4563.0 0.0 2646065.0 14901580.0 85.8 0.5 6.4 0.1 0.0 0.0 14.1 79.4 97 97 97.00 37 1820808049 22.9 25.8 26.6 24.7 0.0 30.5 12.5 bulk 330181 SRR1182138 SRP039090 SRS565447 SRX480564 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 2 (N6) Stomach - RNA-seq "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 2 (N6) Stomach|sex;;male|tissue;;Stomach 194 Young Adult 2 (N6) Stomach 2810639020 14487830 2017-01-20 00:00:00 1821441351 2810639020 14487830 2 14487830 index:0,count:14487830,average:97,stdev:0|index:1,count:14487830,average:97,stdev:0 Young Adult 2 (N6) Stomach - RNA-seq Stanford University 1.52 1.05 0.02 2373668039 3089168215 1775581850 2479916435 130.14 139.67 12742814 9215419 325.971 3113.664 221 68098 70.1 93.96 23906737 8932860 23906737 8932860 89.02 88.24 23906737 11343146 23906737 8389507 58261387 2.45 0.37 0 22.33 0 1.11 0 0.02 0 0.00 0 10.92 0 12742814 0 194 0 188.23 0 1.78 0 0.00 0 1.52 0 0.00 0 220.07 0 1.46 0 53382 0 14487830 0 3235234 0 161346 0 2287 0 0 0 1581383 0 1610 0 0 0 22043 0 4716894 0 10944 0 4751491 0 65.62 0 9507580 0 169591 11226366 66.196708551751 14487830.0 12742814.0 53382.0 3235234.0 161346.0 2287.0 0.0 1581383.0 9507580.0 88.0 0.4 22.3 1.1 0.0 0.0 10.9 65.6 97 97 97.00 37 1405319510 21.6 25.8 28.9 23.7 0.0 24.8 9.6 bulk 330276 SRR1182144 SRP039090 SRS565449 SRX480570 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 3 (Non-N6 Non-N37) Lung - RNA-seq - (rep1) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 3 (Non-N6 Non-N37) Lung|sex;;male|tissue;;Lung 194 Young Adult 3 (Non-N6 Non-N37) Lung 2617693992 13493268 2017-01-20 00:00:00 1954202686 2617693992 13493268 2 13493268 index:0,count:13493268,average:97,stdev:0|index:1,count:13493268,average:97,stdev:0 Young Adult 3 (Non-N6 Non-N37) Lung - RNA-seq - (rep1) Stanford University 0.46 2.09 0.03 2376752033 3468733478 2073876133 3048891877 145.94 147.01 12740571 10844059 274.593 1174.409 201 122572 85.47 97.87 16485152 10889099 16485152 10889099 91.15 91.62 16485152 11613576 16485152 10193320 17904983 0.75 0.36 0 11.97 0 0.03 0 0.01 0 0.00 0 5.54 0 12740571 0 194 0 189.50 0 1.88 0 0.00 0 1.50 0 0.00 0 254.32 0 1.12 0 48447 0 13493268 0 1614883 0 3899 0 785 0 0 0 748013 0 2233 0 0 0 31511 0 6379135 0 15130 0 6428009 0 82.45 0 11125688 0 177335 6695588 37.756720331576 13493268.0 12740571.0 48447.0 1614883.0 3899.0 785.0 0.0 748013.0 11125688.0 94.4 0.4 12.0 0.0 0.0 0.0 5.5 82.5 97 97 97.00 38 1308846996 20.6 27.6 27.2 24.6 0.0 29.6 11.5 bulk 330284 SRR1182145 SRP039090 SRS565449 SRX480571 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 3 (Non-N6 Non-N37) Lung - RNA-seq - (rep2) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 3 (Non-N6 Non-N37) Lung|sex;;male|tissue;;Lung 194 Young Adult 3 (Non-N6 Non-N37) Lung 2021863150 10421975 2017-01-20 00:00:00 1394828848 2021863150 10421975 2 10421975 index:0,count:10421975,average:97,stdev:0|index:1,count:10421975,average:97,stdev:0 Young Adult 3 (Non-N6 Non-N37) Lung - RNA-seq - (rep2) Stanford University 13.93 1.18 0.04 1283910292 1787622126 1147258517 1684496694 139.23 146.83 7796791 6949657 235.152 1212.708 256 36306 73.13 83.38 12883014 5702123 12883014 5702123 74.21 80.53 12883014 5786071 12883014 5506846 113329660 8.83 4.61 0 9.19 0 0.33 0 0.03 0 0.00 0 24.83 0 7796791 0 194 0 185.40 0 1.41 0 0.01 0 1.79 0 0.01 0 240.51 0 1.08 0 480305 0 10421975 0 958199 0 34829 0 2656 0 0 0 2587699 0 780 0 0 0 11171 0 1747536 0 18296 0 1777783 0 65.62 0 6838592 0 105904 1703530 16.085605831697 10421975.0 7796791.0 480305.0 958199.0 34829.0 2656.0 0.0 2587699.0 6838592.0 74.8 4.6 9.2 0.3 0.0 0.0 24.8 65.6 97 97 97.00 38 1010931575 28.2 21.8 24.5 25.4 0.1 27.7 10.5 bulk 330374 SRR1182150 SRP039090 SRS565450 SRX480576 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 4 (Non-N6 Non-N37) Liver - RNA-seq - (rep1) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 4 (Non-N6 Non-N37) Liver|sex;;male|tissue;;Liver 194 Young Adult 4 (Non-N6 Non-N37) Liver 1642467244 8466326 2017-01-20 00:00:00 1174422273 1642467244 8466326 2 8466326 index:0,count:8466326,average:97,stdev:0|index:1,count:8466326,average:97,stdev:0 Young Adult 4 (Non-N6 Non-N37) Liver - RNA-seq - (rep1) Stanford University 3.8 1.39 0.03 1287749381 1767754940 1179750800 1657290292 137.27 140.48 7094010 5927389 270.807 1202.953 208 54162 86.84 94.8 9163016 6160745 9163016 6160745 86.01 88.5 9163016 6101457 9163016 5751377 29213501 2.27 0.36 0 7.03 0 0.05 0 0.03 0 0.00 0 16.13 0 7094010 0 194 0 187.62 0 1.83 0 0.00 0 1.60 0 0.00 0 211.66 0 1.63 0 30678 0 8466326 0 595409 0 4435 0 2340 0 0 0 1365541 0 1055 0 0 0 16660 0 3311729 0 7133 0 3336577 0 76.76 0 6498601 0 143257 3612170 25.214614294589 8466326.0 7094010.0 30678.0 595409.0 4435.0 2340.0 0.0 1365541.0 6498601.0 83.8 0.4 7.0 0.1 0.0 0.0 16.1 76.8 97 97 97.00 38 821233622 21.5 27.0 27.1 24.3 0.0 21.0 7.5 bulk 660760 SRR1182151 SRP039090 SRS565450 SRX480577 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 4 (Non-N6 Non-N37) Liver - RNA-seq - (rep2) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 4 (Non-N6 Non-N37) Liver|sex;;male|tissue;;Liver 194 Young Adult 4 (Non-N6 Non-N37) Liver 4665805924 24050546 2017-01-20 00:00:00 3136371420 4665805924 24050546 2 24050546 index:0,count:24050546,average:97,stdev:0|index:1,count:24050546,average:97,stdev:0 Young Adult 4 (Non-N6 Non-N37) Liver - RNA-seq - (rep2) Stanford University 32.21 0.95 0.05 2747605088 4164848734 2531039128 3965297421 151.58 156.67 17551101 16120578 215.027 868.255 103 86646 75.0 82.56 24376629 13164065 24376629 13164065 76.15 79.81 24376629 13365841 24376629 12726095 274487586 9.99 5.28 0 6.68 0 0.22 0 0.02 0 0.00 0 26.78 0 17551101 0 194 0 184.31 0 1.47 0 0.02 0 1.72 0 0.01 0 295.50 0 0.99 0 1270536 0 24050546 0 1605842 0 52156 0 5863 0 0 0 6441426 0 1166 0 0 0 17005 0 2603738 0 79242 0 2701151 0 66.30 0 15945259 0 118183 2520309 21.325478283679 24050546.0 17551101.0 1270536.0 1605842.0 52156.0 5863.0 0.0 6441426.0 15945259.0 73.0 5.3 6.7 0.2 0.0 0.0 26.8 66.3 97 97 97.00 38 2332902962 28.0 22.1 23.9 25.9 0.1 30.0 11.3 bulk 660826 SRR1182155 SRP039090 SRS565451 SRX480581 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 5 (Non-N6 Non-N37) Small Intestine - RNA-seq - (rep1) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 5 (Non-N6 Non-N37) Small Intestine|sex;;male|tissue;;Small Intestine 194 Young Adult 5 (Non-N6 Non-N37) Small Intestine 2298365724 11847246 2017-01-20 00:00:00 1626578899 2298365724 11847246 2 11847246 index:0,count:11847246,average:97,stdev:0|index:1,count:11847246,average:97,stdev:0 Young Adult 5 (Non-N6 Non-N37) Small Intestine - RNA-seq - (rep1) Stanford University 3.84 1.85 0.03 1729374202 2412866959 1601698706 2276606898 139.52 142.14 9607217 8184344 260.232 1445.094 193 68685 87.49 94.49 11991523 8405565 11991523 8405565 86.13 88.31 11991523 8274994 11991523 7855442 41320227 2.39 0.38 0 6.01 0 0.06 0 0.05 0 0.00 0 18.80 0 9607217 0 194 0 187.67 0 1.85 0 0.00 0 1.58 0 0.00 0 247.97 0 1.61 0 44561 0 11847246 0 711864 0 6830 0 5340 0 0 0 2227859 0 1678 0 0 0 22807 0 4473434 0 10309 0 4508228 0 75.08 0 8895353 0 169091 4717803 27.900970483349 11847246.0 9607217.0 44561.0 711864.0 6830.0 5340.0 0.0 2227859.0 8895353.0 81.1 0.4 6.0 0.1 0.0 0.0 18.8 75.1 97 97 97.00 38 1149182862 21.7 27.1 27.4 23.9 0.0 20.7 7.4 bulk 660840 SRR1182156 SRP039090 SRS565451 SRX480582 SRA143022 Stanford University Li Lab A Quantitative Mammalian Atlas of A-to-I RNA Editing Human RNA-seq/exome-seq and mouse RNA-seq libraries were generated to identify A-to-I RNA editing events. RNA editing levels at >10,000 exonic sites in >400 human and mouse samples were profiled by mmPCR-seq. Young Adult 5 (Non-N6 Non-N37) Small Intestine - RNA-seq - (rep2) "The general Illumina mRNA-seq library preparation workflow was followed with some modifications. In particular, these libraries do not follow the standard 6bp Illumina barcodes. Instead, custom 3bp barcodes were inserted at the ligated end of the adapters. Hence, during the library amplication step, common forward and reverse primers were used for all libraries, since the barcodes had already been added during ligation." RNA-Seq TRANSCRIPTOMIC cDNA paired Illumina HiSeq 2000 age;;Around 25 years|biomaterial_provider;;BioChain|BioSampleModel;;Human|isolate;;Not Collected|label;;Young Adult 5 (Non-N6 Non-N37) Small Intestine|sex;;male|tissue;;Small Intestine 194 Young Adult 5 (Non-N6 Non-N37) Small Intestine 3591049222 18510563 2017-01-20 00:00:00 2433959185 3591049222 18510563 2 18510563 index:0,count:18510563,average:97,stdev:0|index:1,count:18510563,average:97,stdev:0 Young Adult 5 (Non-N6 Non-N37) Small Intestine - RNA-seq - (rep2) Stanford University 32.9 1.16 0.07 1988653990 3038741365 1847603652 2908102963 152.8 157.4 12794141 11946389 211.798 900.287 97 67767 73.54 80.2 17278272 9408896 17278272 9408896 74.54 77.73 17278272 9536741 17278272 9118740 235179165 11.83 5.02 0 5.74 0 0.20 0 0.09 0 0.00 0 30.60 0 12794141 0 194 0 184.25 0 1.37 0 0.02 0 1.81 0 0.01 0 12.44 0 0.98 0 928806 0 18510563 0 1062382 0 36269 0 15934 0 0 0 5664219 0 976 0 0 0 12530 0 1634530 0 48313 0 1696349 0 63.38 0 11731759 0 116859 1545716 13.227188320968 18510563.0 12794141.0 928806.0 1062382.0 36269.0 15934.0 0.0 5664219.0 11731759.0 69.1 5.0 5.7 0.2 0.1 0.0 30.6 63.4 97 97 97.00 38 1795524611 28.9 21.9 23.8 25.2 0.1 29.4 11.4 bulk 219479 SRR2042655 SRP058773 SRS947595 SRX1040931 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697224: q48_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;5 GEO Accession;;GSM1697224 GSM1697224 q48_0 79578150 1591563 2015-07-16 16:21:14 46546165 79578150 1591563 2 1591563 index:0,count:1591563,average:25,stdev:0|index:1,count:1591563,average:25,stdev:0 GSM1697224_r1 GEO 1.7 2.38 2.66 38051921 27826579 33309886 25316150 73.13 76.0 777729 705189 227.602 6332.389 221 3689 15.04 17.23 1108684 116936 1108684 116936 16.95 16.67 1108684 131831 1108684 113129 12662775 33.28 2.08 0 6.21 0 1.56 0 2.20 0 0.00 0 47.37 0 777729 0 50 0 49.13 0 1.15 0 0.00 0 1.01 0 0.00 0 197.57 0 0.74 0 33065 0 1591563 0 98889 0 24880 0 35013 0 0 0 753941 0 1 0 0 0 7 0 545 0 161 0 714 0 42.65 0 678840 0 144 207 1.437500000000 1591563.0 777729.0 33065.0 98889.0 24880.0 35013.0 0.0 753941.0 678840.0 48.9 2.1 6.2 1.6 2.2 0.0 47.4 42.7 25 25 25.00 7 39789075 28.3 20.2 19.9 31.6 0.0 31.2 17.6 smartseq 219483 SRR2042656 SRP058773 SRS947567 SRX1040932 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697225: q49_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;32 GEO Accession;;GSM1697225 GSM1697225 q49_0 189777600 3795552 2015-07-16 16:21:14 110612200 189777600 3795552 2 3795552 index:0,count:3795552,average:25,stdev:0|index:1,count:3795552,average:25,stdev:0 GSM1697225_r1 GEO 6.4 2.02 0.19 106400388 134359042 85704142 114786731 126.28 133.93 2171711 1822924 255.045 3628.450 218 8394 59.45 73.84 3918021 1291003 3918021 1291003 66.79 70.45 3918021 1450503 3918021 1231824 11880336 11.17 1.38 0 11.15 0 0.55 0 0.69 0 0.00 0 41.54 0 2171711 0 50 0 49.08 0 1.16 0 0.00 0 1.01 0 0.00 0 77.20 0 0.82 0 52235 0 3795552 0 423256 0 20928 0 26122 0 0 0 1576791 0 5 0 0 0 26 0 5825 0 283 0 6139 0 46.07 0 1748455 0 1088 3134 2.880514705882 3795552.0 2171711.0 52235.0 423256.0 20928.0 26122.0 0.0 1576791.0 1748455.0 57.2 1.4 11.2 0.6 0.7 0.0 41.5 46.1 25 25 25.00 7 94888800 27.6 21.8 21.8 28.7 0.0 31.3 17.6 smartseq 438447 SRR2042615 SRP058773 SRS947440 SRX1040892 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697185: q9_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;90 GEO Accession;;GSM1697185 GSM1697185 q9_0 275954750 5519095 2015-07-16 16:21:14 160707981 275954750 5519095 2 5519095 index:0,count:5519095,average:25,stdev:0|index:1,count:5519095,average:25,stdev:0 GSM1697185_r1 GEO 4.79 2.2 0.2 154514039 182777567 136160074 165122550 118.29 121.27 3146607 2819910 245.721 3234.415 227 13370 43.22 49.09 4593988 1359979 4593988 1359979 47.4 47.21 4593988 1491540 4593988 1307844 43911323 28.42 1.24 0 6.81 0 0.36 0 0.72 0 0.00 0 41.90 0 3146607 0 50 0 49.19 0 1.21 0 0.00 0 1.01 0 0.00 0 251.50 0 0.78 0 68439 0 5519095 0 376094 0 20073 0 39833 0 0 0 2312582 0 8 0 0 0 45 0 4961 0 382 0 5396 0 50.20 0 2770513 0 1017 2394 2.353982300885 5519095.0 3146607.0 68439.0 376094.0 20073.0 39833.0 0.0 2312582.0 2770513.0 57.0 1.2 6.8 0.4 0.7 0.0 41.9 50.2 25 25 25.00 7 137977375 28.7 21.4 21.1 28.8 0.0 31.0 17.3 smartseq 438455 SRR2042616 SRP058773 SRS947439 SRX1040893 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697186: q10_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;61 GEO Accession;;GSM1697186 GSM1697186 q10_0 224701450 4494029 2015-07-16 16:21:14 131119189 224701450 4494029 2 4494029 index:0,count:4494029,average:25,stdev:0|index:1,count:4494029,average:25,stdev:0 GSM1697186_r1 GEO 2.71 2.13 0.51 118115440 131280469 103292716 118483376 111.15 114.71 2413103 2175345 236.044 3994.870 207 10574 37.26 42.66 3612652 899025 3612652 899025 41.01 40.89 3612652 989607 3612652 861863 35827700 30.33 1.34 0 6.80 0 0.66 0 0.95 0 0.00 0 44.70 0 2413103 0 50 0 49.09 0 1.23 0 0.00 0 1.02 0 0.00 0 227.87 0 0.79 0 60385 0 4494029 0 305571 0 29697 0 42615 0 0 0 2008614 0 2 0 0 0 44 0 3433 0 370 0 3849 0 46.90 0 2107532 0 716 1612 2.251396648045 4494029.0 2413103.0 60385.0 305571.0 29697.0 42615.0 0.0 2008614.0 2107532.0 53.7 1.3 6.8 0.7 0.9 0.0 44.7 46.9 25 25 25.00 7 112350725 28.8 21.0 21.1 29.1 0.0 31.0 17.2 smartseq 438559 SRR2042623 SRP058773 SRS947432 SRX1040900 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697193: q17_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;77 GEO Accession;;GSM1697193 GSM1697193 q17_0 55214350 1104287 2015-07-16 16:21:14 32134477 55214350 1104287 2 1104287 index:0,count:1104287,average:25,stdev:0|index:1,count:1104287,average:25,stdev:0 GSM1697193_r1 GEO 1.38 2.72 1.99 21195928 15424178 18164131 13678312 72.77 75.3 435568 388407 227.123 8847.306 224 1946 12.16 14.26 685440 52975 685440 52975 14.69 13.53 685440 63995 685440 50251 7236990 34.14 1.78 0 5.80 0 1.31 0 2.91 0 0.00 0 56.33 0 435568 0 50 0 49.00 0 1.18 0 0.00 0 1.00 0 0.00 0 147.24 0 0.76 0 19668 0 1104287 0 64053 0 14500 0 32179 0 0 0 622040 0 3 0 0 0 6 0 273 0 117 0 399 0 33.64 0 371515 0 57 89 1.561403508772 1104287.0 435568.0 19668.0 64053.0 14500.0 32179.0 0.0 622040.0 371515.0 39.4 1.8 5.8 1.3 2.9 0.0 56.3 33.6 25 25 25.00 7 27607175 26.4 18.5 18.5 36.6 0.0 31.5 17.8 smartseq 438567 SRR2042624 SRP058773 SRS947430 SRX1040901 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697194: q18_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;20 GEO Accession;;GSM1697194 GSM1697194 q18_0 115186900 2303738 2015-07-16 16:21:14 67306291 115186900 2303738 2 2303738 index:0,count:2303738,average:25,stdev:0|index:1,count:2303738,average:25,stdev:0 GSM1697194_r1 GEO 3.46 2.19 1.21 60129722 67958739 52998845 62264977 113.02 117.48 1226798 1100940 232.353 4238.193 207 5540 41.82 47.53 1785230 513088 1785230 513088 44.94 45.93 1785230 551343 1785230 495821 16459268 27.37 1.35 0 6.40 0 0.82 0 1.05 0 0.00 0 44.88 0 1226798 0 50 0 49.17 0 1.19 0 0.00 0 1.04 0 0.00 0 180.29 0 0.79 0 30988 0 2303738 0 147375 0 18843 0 24218 0 0 0 1033879 0 2 0 0 0 10 0 1622 0 158 0 1792 0 46.86 0 1079423 0 397 689 1.735516372796 2303738.0 1226798.0 30988.0 147375.0 18843.0 24218.0 0.0 1033879.0 1079423.0 53.3 1.3 6.4 0.8 1.1 0.0 44.9 46.9 25 25 25.00 7 57593450 28.9 20.5 20.3 30.2 0.0 31.2 17.5 smartseq 438575 SRR2042625 SRP058773 SRS947431 SRX1040902 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697195: q19_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;43 GEO Accession;;GSM1697195 GSM1697195 q19_0 69745950 1394919 2015-07-16 16:21:14 40879418 69745950 1394919 2 1394919 index:0,count:1394919,average:25,stdev:0|index:1,count:1394919,average:25,stdev:0 GSM1697195_r1 GEO 2.39 1.19 2.0 30944246 23706316 22571345 19303566 76.61 85.52 635635 552702 221.826 7746.402 207 2892 18.27 25.13 1468642 116143 1468642 116143 24.24 24.07 1468642 154081 1468642 111258 7661500 24.76 1.85 0 12.43 0 2.47 0 2.03 0 0.00 0 49.94 0 635635 0 50 0 48.97 0 1.18 0 0.00 0 1.00 0 0.00 0 33.48 0 0.84 0 25755 0 1394919 0 173453 0 34422 0 28251 0 0 0 696611 0 0 0 0 0 10 0 623 0 157 0 790 0 33.13 0 462182 0 185 283 1.529729729730 1394919.0 635635.0 25755.0 173453.0 34422.0 28251.0 0.0 696611.0 462182.0 45.6 1.8 12.4 2.5 2.0 0.0 49.9 33.1 25 25 25.00 7 34872975 27.7 21.1 21.1 30.1 0.0 31.3 17.6 smartseq 438839 SRR2042646 SRP058773 SRS947400 SRX1040923 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697216: q40_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;48 GEO Accession;;GSM1697216 GSM1697216 q40_0 63443550 1268871 2015-07-16 16:21:14 37292313 63443550 1268871 2 1268871 index:0,count:1268871,average:25,stdev:0|index:1,count:1268871,average:25,stdev:0 GSM1697216_r1 GEO 0.94 2.7 0.44 29817490 32275638 27691784 30474868 108.24 110.05 607750 563855 236.831 4208.044 218 2802 32.02 34.54 764942 194575 764942 194575 33.35 33.36 764942 202671 764942 187964 11069042 37.12 1.37 0 3.50 0 0.30 0 0.88 0 0.00 0 50.92 0 607750 0 50 0 49.25 0 1.19 0 0.00 0 1.01 0 0.00 0 46.61 0 0.76 0 17441 0 1268871 0 44365 0 3809 0 11213 0 0 0 646099 0 0 0 0 0 9 0 668 0 120 0 797 0 44.40 0 563385 0 169 240 1.420118343195 1268871.0 607750.0 17441.0 44365.0 3809.0 11213.0 0.0 646099.0 563385.0 47.9 1.4 3.5 0.3 0.9 0.0 50.9 44.4 25 25 25.00 7 31721775 29.4 19.5 19.9 31.2 0.0 31.1 17.3 smartseq 438847 SRR2042647 SRP058773 SRS947398 SRX1040924 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697217: q41_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;6 GEO Accession;;GSM1697217 GSM1697217 q41_0 59074400 1181488 2015-07-16 16:21:14 34625537 59074400 1181488 2 1181488 index:0,count:1181488,average:25,stdev:0|index:1,count:1181488,average:25,stdev:0 GSM1697217_r1 GEO 4.98 1.69 0.25 30852953 39701702 22208674 31609067 128.68 142.33 631854 526400 237.110 4295.935 217 2621 66.53 92.47 1393545 420385 1393545 420385 78.65 88.34 1393545 496941 1393545 401616 767173 2.49 1.37 0 15.00 0 0.43 0 1.08 0 0.00 0 45.01 0 631854 0 50 0 48.93 0 1.19 0 0.00 0 1.01 0 0.00 0 170.13 0 0.83 0 16205 0 1181488 0 177251 0 5061 0 12777 0 0 0 531796 0 0 0 0 0 10 0 1667 0 98 0 1775 0 38.48 0 454603 0 364 780 2.142857142857 1181488.0 631854.0 16205.0 177251.0 5061.0 12777.0 0.0 531796.0 454603.0 53.5 1.4 15.0 0.4 1.1 0.0 45.0 38.5 25 25 25.00 7 29537200 26.7 20.8 21.1 31.3 0.0 31.1 17.3 smartseq 438853 SRR2042648 SRP058773 SRS947399 SRX1040925 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697218: q42_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;69 GEO Accession;;GSM1697218 GSM1697218 q42_0 304449700 6088994 2015-07-16 16:21:14 177529583 304449700 6088994 2 6088994 index:0,count:6088994,average:25,stdev:0|index:1,count:6088994,average:25,stdev:0 GSM1697218_r1 GEO 5.14 1.72 0.24 170571051 204487027 138247150 175740782 119.88 127.12 3486076 2985171 252.638 3459.614 207 13682 51.46 63.52 6407587 1793825 6407587 1793825 58.75 60.96 6407587 2048153 6407587 1721508 30339893 17.79 1.34 0 10.88 0 0.69 0 0.63 0 0.00 0 41.43 0 3486076 0 50 0 49.02 0 1.19 0 0.00 0 1.01 0 0.00 0 257.89 0 0.82 0 81527 0 6088994 0 662241 0 42140 0 38209 0 0 0 2522569 0 3 0 0 0 50 0 7642 0 431 0 8126 0 46.38 0 2823835 0 1420 4036 2.842253521127 6088994.0 3486076.0 81527.0 662241.0 42140.0 38209.0 0.0 2522569.0 2823835.0 57.3 1.3 10.9 0.7 0.6 0.0 41.4 46.4 25 25 25.00 7 152224850 28.1 22.0 22.0 27.9 0.0 31.1 17.3 smartseq 438861 SRR2042649 SRP058773 SRS947397 SRX1040926 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697219: q43_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;73 GEO Accession;;GSM1697219 GSM1697219 q43_0 207776700 4155534 2015-07-16 16:21:14 121047347 207776700 4155534 2 4155534 index:0,count:4155534,average:25,stdev:0|index:1,count:4155534,average:25,stdev:0 GSM1697219_r1 GEO 2.28 2.71 0.57 111144277 117609308 99131848 107487122 105.82 108.43 2265340 2070747 244.269 3930.259 218 9730 33.47 37.58 3196534 758291 3196534 758291 36.2 35.95 3196534 820077 3196534 725329 34284956 30.85 1.41 0 5.96 0 0.78 0 1.22 0 0.00 0 43.49 0 2265340 0 50 0 49.20 0 1.12 0 0.00 0 1.01 0 0.00 0 241.29 0 0.77 0 58638 0 4155534 0 247697 0 32308 0 50565 0 0 0 1807321 0 1 0 0 0 14 0 2606 0 379 0 3000 0 48.55 0 2017643 0 565 1229 2.175221238938 4155534.0 2265340.0 58638.0 247697.0 32308.0 50565.0 0.0 1807321.0 2017643.0 54.5 1.4 6.0 0.8 1.2 0.0 43.5 48.6 25 25 25.00 7 103888350 28.5 20.8 20.9 29.8 0.0 31.2 17.5 smartseq 438917 SRR2042650 SRP058773 SRS947396 SRX1040927 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697220: q44_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;10 GEO Accession;;GSM1697220 GSM1697220 q44_0 63014850 1260297 2015-07-16 16:21:14 36915249 63014850 1260297 2 1260297 index:0,count:1260297,average:25,stdev:0|index:1,count:1260297,average:25,stdev:0 GSM1697220_r1 GEO 3.44 1.97 1.68 29271434 24926124 24589655 21826605 85.16 88.76 599627 535590 217.307 6421.584 207 2859 23.55 28.12 973117 141190 973117 141190 27.3 26.81 973117 163683 973117 134649 8397992 28.69 1.81 0 7.73 0 1.53 0 2.05 0 0.00 0 48.84 0 599627 0 50 0 49.06 0 1.19 0 0.00 0 1.04 0 0.00 0 189.04 0 0.77 0 22809 0 1260297 0 97444 0 19292 0 25823 0 0 0 615555 0 1 0 0 0 8 0 613 0 130 0 752 0 39.85 0 502183 0 144 231 1.604166666667 1260297.0 599627.0 22809.0 97444.0 19292.0 25823.0 0.0 615555.0 502183.0 47.6 1.8 7.7 1.5 2.0 0.0 48.8 39.8 25 25 25.00 7 31507425 27.6 20.4 20.2 31.7 0.0 31.3 17.6 smartseq 438933 SRR2042652 SRP058773 SRS947395 SRX1040928 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697221: q45_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;31 GEO Accession;;GSM1697221 GSM1697221 q45_0 76798300 1535966 2015-07-16 16:21:14 44781369 76798300 1535966 2 1535966 index:0,count:1535966,average:25,stdev:0|index:1,count:1535966,average:25,stdev:0 GSM1697221_r1 GEO 2.61 2.33 2.96 31984326 23628904 27051818 21165363 73.88 78.24 657279 573501 230.251 8499.770 220 2846 16.24 19.29 1003269 106765 1003269 106765 18.22 18.43 1003269 119770 1003269 102001 10075718 31.50 1.95 0 6.76 0 1.99 0 2.24 0 0.00 0 52.98 0 657279 0 50 0 48.99 0 1.14 0 0.00 0 1.03 0 0.00 0 153.60 0 0.80 0 29953 0 1535966 0 103827 0 30578 0 34341 0 0 0 813768 0 1 0 0 0 9 0 555 0 171 0 736 0 36.03 0 553452 0 97 185 1.907216494845 1535966.0 657279.0 29953.0 103827.0 30578.0 34341.0 0.0 813768.0 553452.0 42.8 2.0 6.8 2.0 2.2 0.0 53.0 36.0 25 25 25.00 7 38399150 27.6 19.8 19.7 32.9 0.0 31.4 17.6 smartseq 438941 SRR2042653 SRP058773 SRS947394 SRX1040929 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697222: q46_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;9 GEO Accession;;GSM1697222 GSM1697222 q46_0 32070900 641418 2015-07-16 16:21:14 18890384 32070900 641418 2 641418 index:0,count:641418,average:25,stdev:0|index:1,count:641418,average:25,stdev:0 GSM1697222_r1 GEO 2.02 2.12 2.92 14478988 10477449 11946726 9241604 72.36 77.36 296201 262157 245.836 6666.575 198 1252 15.44 18.77 501479 45719 501479 45719 18.48 18.09 501479 54751 501479 44047 4370437 30.18 1.65 0 8.21 0 2.39 0 2.50 0 0.00 0 48.93 0 296201 0 50 0 49.12 0 1.10 0 0.00 0 1.00 0 0.00 0 76.97 0 0.78 0 10560 0 641418 0 52677 0 15312 0 16029 0 0 0 313876 0 0 0 0 0 1 0 241 0 46 0 288 0 37.97 0 243524 0 63 83 1.317460317460 641418.0 296201.0 10560.0 52677.0 15312.0 16029.0 0.0 313876.0 243524.0 46.2 1.6 8.2 2.4 2.5 0.0 48.9 38.0 25 25 25.00 7 16035450 28.3 19.6 19.3 32.7 0.0 31.3 17.7 smartseq 438949 SRR2042654 SRP058773 SRS947393 SRX1040930 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697223: q47_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;66 GEO Accession;;GSM1697223 GSM1697223 q47_0 82437050 1648741 2015-07-16 16:21:14 48124411 82437050 1648741 2 1648741 index:0,count:1648741,average:25,stdev:0|index:1,count:1648741,average:25,stdev:0 GSM1697223_r1 GEO 0.59 2.29 3.68 32854247 20557648 28622195 19000460 62.57 66.38 676460 596806 225.278 9602.845 199 2927 6.62 7.64 958098 44787 958098 44787 7.4 7.46 958098 50087 958098 43768 11887812 36.18 1.94 0 5.45 0 2.07 0 2.26 0 0.00 0 54.64 0 676460 0 50 0 48.91 0 1.12 0 0.00 0 1.04 0 0.00 0 237.42 0 0.78 0 31999 0 1648741 0 89931 0 34110 0 37302 0 0 0 900869 0 1 0 0 0 14 0 344 0 221 0 580 0 35.57 0 586529 0 69 97 1.405797101449 1648741.0 676460.0 31999.0 89931.0 34110.0 37302.0 0.0 900869.0 586529.0 41.0 1.9 5.5 2.1 2.3 0.0 54.6 35.6 25 25 25.00 7 41218525 28.5 19.6 19.6 32.3 0.0 31.2 17.4 smartseq 438972 SRR2042657 SRP058773 SRS947594 SRX1040933 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697226: q50_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;81 GEO Accession;;GSM1697226 GSM1697226 q50_0 88763750 1775275 2015-07-16 16:21:14 51919952 88763750 1775275 2 1775275 index:0,count:1775275,average:25,stdev:0|index:1,count:1775275,average:25,stdev:0 GSM1697226_r1 GEO 6.39 1.77 0.22 45778801 59987460 33638562 48673228 131.04 144.69 937811 759684 252.987 4136.342 218 3584 68.82 93.66 2025663 645432 2025663 645432 80.3 89.82 2025663 753025 2025663 618954 899379 1.96 1.31 0 14.01 0 0.36 0 0.73 0 0.00 0 46.09 0 937811 0 50 0 48.91 0 1.26 0 0.00 0 1.04 0 0.00 0 41.23 0 0.87 0 23272 0 1775275 0 248709 0 6351 0 12935 0 0 0 818178 0 1 0 0 0 24 0 2898 0 122 0 3045 0 38.82 0 689102 0 544 1555 2.858455882353 1775275.0 937811.0 23272.0 248709.0 6351.0 12935.0 0.0 818178.0 689102.0 52.8 1.3 14.0 0.4 0.7 0.0 46.1 38.8 25 25 25.00 7 44381875 26.7 21.6 21.7 30.0 0.0 31.3 17.6 smartseq 438980 SRR2042658 SRP058773 SRS947590 SRX1040934 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697227: q51_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;67 GEO Accession;;GSM1697227 GSM1697227 q51_0 237653400 4753068 2015-07-16 16:21:14 138602638 237653400 4753068 2 4753068 index:0,count:4753068,average:25,stdev:0|index:1,count:4753068,average:25,stdev:0 GSM1697227_r1 GEO 1.77 2.36 1.0 126029788 120204471 115770333 112553919 95.38 97.22 2570004 2396665 243.562 4142.929 218 11610 20.47 22.32 3248792 526074 3248792 526074 21.75 21.51 3248792 558966 3248792 507005 49507625 39.28 1.40 0 4.48 0 0.96 0 1.34 0 0.00 0 43.63 0 2570004 0 50 0 49.18 0 1.17 0 0.00 0 1.00 0 0.00 0 244.44 0 0.75 0 66651 0 4753068 0 213023 0 45660 0 63821 0 0 0 2073583 0 2 0 0 0 19 0 1759 0 374 0 2154 0 49.59 0 2356981 0 388 775 1.997422680412 4753068.0 2570004.0 66651.0 213023.0 45660.0 63821.0 0.0 2073583.0 2356981.0 54.1 1.4 4.5 1.0 1.3 0.0 43.6 49.6 25 25 25.00 7 118826700 29.4 20.4 20.4 29.8 0.0 31.1 17.4 smartseq 438988 SRR2042659 SRP058773 SRS947592 SRX1040935 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697228: q52_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;50 GEO Accession;;GSM1697228 GSM1697228 q52_0 54265000 1085300 2015-07-16 16:21:14 31820877 54265000 1085300 2 1085300 index:0,count:1085300,average:25,stdev:0|index:1,count:1085300,average:25,stdev:0 GSM1697228_r1 GEO 1.09 2.09 2.69 22490726 16807394 18915166 15021874 74.73 79.42 461521 399979 234.846 8336.312 221 2114 17.7 21.13 732648 81707 732648 81707 20.31 20.43 732648 93734 732648 78999 6915174 30.75 1.84 0 6.90 0 1.96 0 2.24 0 0.00 0 53.28 0 461521 0 50 0 49.01 0 1.12 0 0.00 0 1.04 0 0.00 0 139.54 0 0.82 0 19984 0 1085300 0 74911 0 21267 0 24285 0 0 0 578227 0 0 0 0 0 4 0 465 0 112 0 581 0 35.62 0 386610 0 144 190 1.319444444444 1085300.0 461521.0 19984.0 74911.0 21267.0 24285.0 0.0 578227.0 386610.0 42.5 1.8 6.9 2.0 2.2 0.0 53.3 35.6 25 25 25.00 7 27132500 27.9 20.2 19.7 32.2 0.0 31.3 17.8 smartseq 876664 SRR2042607 SRP058773 SRS947447 SRX1040883 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697177: q1_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;22 GEO Accession;;GSM1697177 GSM1697177 q1_0 103363000 2067260 2015-07-16 16:21:14 60411378 103363000 2067260 2 2067260 index:0,count:2067260,average:25,stdev:0|index:1,count:2067260,average:25,stdev:0 GSM1697177_r1 GEO 5.64 1.71 1.45 50481425 54589350 39870775 45820929 108.14 114.92 1034275 870598 232.194 5520.274 207 4358 47.63 60.41 1883062 492600 1883062 492600 54.93 57.98 1883062 568110 1883062 472782 7148457 14.16 1.59 0 10.59 0 1.46 0 1.22 0 0.00 0 47.29 0 1034275 0 50 0 48.96 0 1.25 0 0.00 0 1.02 0 0.00 0 140.42 0 0.86 0 32866 0 2067260 0 218878 0 30179 0 25245 0 0 0 977561 0 2 0 0 0 12 0 1965 0 166 0 2145 0 39.44 0 815397 0 517 961 1.858800773694 2067260.0 1034275.0 32866.0 218878.0 30179.0 25245.0 0.0 977561.0 815397.0 50.0 1.6 10.6 1.5 1.2 0.0 47.3 39.4 25 25 25.00 7 51681500 27.9 21.4 21.3 29.3 0.0 31.3 17.6 smartseq 876681 SRR2042608 SRP058773 SRS947449 SRX1040884 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697178: q2_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;91 GEO Accession;;GSM1697178 GSM1697178 q2_0 111543800 2230876 2015-07-16 16:21:14 65319225 111543800 2230876 2 2230876 index:0,count:2230876,average:25,stdev:0|index:1,count:2230876,average:25,stdev:0 GSM1697178_r1 GEO 2.77 2.48 0.55 61258675 67830817 53356565 60980077 110.73 114.29 1250300 1116897 228.390 3625.211 215 5505 38.26 43.98 1854331 478418 1854331 478418 41.95 42.05 1854331 524516 1854331 457396 17629819 28.78 1.37 0 7.29 0 0.77 0 1.16 0 0.00 0 42.02 0 1250300 0 50 0 49.15 0 1.08 0 0.00 0 1.03 0 0.00 0 205.93 0 0.78 0 30541 0 2230876 0 162613 0 17218 0 25882 0 0 0 937476 0 3 0 0 0 19 0 1939 0 168 0 2129 0 48.76 0 1087687 0 438 913 2.084474885845 2230876.0 1250300.0 30541.0 162613.0 17218.0 25882.0 0.0 937476.0 1087687.0 56.0 1.4 7.3 0.8 1.2 0.0 42.0 48.8 25 25 25.00 7 55771900 28.2 21.7 21.5 28.6 0.0 31.1 17.4 smartseq 876698 SRR2042609 SRP058773 SRS947448 SRX1040885 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697179: q3_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;60 GEO Accession;;GSM1697179 GSM1697179 q3_0 32126250 642525 2015-07-16 16:21:14 18844474 32126250 642525 2 642525 index:0,count:642525,average:25,stdev:0|index:1,count:642525,average:25,stdev:0 GSM1697179_r1 GEO 0.54 2.08 2.16 11654253 8175958 9342553 6937459 70.15 74.26 240544 207709 207.945 10745.506 194 1136 11.76 14.73 428692 28281 428692 28281 15.71 13.98 428692 37794 428692 26841 3792460 32.54 1.89 0 7.56 0 1.55 0 2.84 0 0.00 0 58.17 0 240544 0 50 0 48.86 0 1.06 0 0.00 0 1.00 0 0.00 0 72.28 0 0.81 0 12138 0 642525 0 48600 0 9946 0 18264 0 0 0 373771 0 0 0 0 0 4 0 250 0 84 0 338 0 29.87 0 191944 0 61 86 1.409836065574 642525.0 240544.0 12138.0 48600.0 9946.0 18264.0 0.0 373771.0 191944.0 37.4 1.9 7.6 1.5 2.8 0.0 58.2 29.9 25 25 25.00 7 16063125 26.9 18.8 18.6 35.7 0.0 31.4 17.8 smartseq 876809 SRR2042610 SRP058773 SRS947444 SRX1040887 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697180: q4_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;41 GEO Accession;;GSM1697180 GSM1697180 q4_0 20487750 409755 2015-07-16 16:21:14 12064383 20487750 409755 2 409755 index:0,count:409755,average:25,stdev:0|index:1,count:409755,average:25,stdev:0 GSM1697180_r1 GEO 0.25 0.98 0.23 8871829 9497126 4747162 6572338 107.05 138.45 182838 151364 235.296 6611.953 207 783 48.25 90.02 651164 88213 651164 88213 65.1 85.09 651164 119030 651164 83386 251623 2.84 1.36 0 20.71 0 0.67 0 1.92 0 0.00 0 52.79 0 182838 0 50 0 48.65 0 1.18 0 0.00 0 1.00 0 0.00 0 54.63 0 0.85 0 5577 0 409755 0 84841 0 2729 0 7885 0 0 0 216303 0 0 0 0 0 1 0 403 0 38 0 442 0 23.92 0 97997 0 100 178 1.780000000000 409755.0 182838.0 5577.0 84841.0 2729.0 7885.0 0.0 216303.0 97997.0 44.6 1.4 20.7 0.7 1.9 0.0 52.8 23.9 25 25 25.00 7 10243875 24.6 19.8 20.2 35.4 0.0 31.5 17.7 smartseq 876825 SRR2042611 SRP058773 SRS947445 SRX1040888 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697181: q5_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;34 GEO Accession;;GSM1697181 GSM1697181 q5_0 258227300 5164546 2015-07-16 16:21:14 150325742 258227300 5164546 2 5164546 index:0,count:5164546,average:25,stdev:0|index:1,count:5164546,average:25,stdev:0 GSM1697181_r1 GEO 6.71 1.84 0.19 153503668 204235654 117759712 168715772 133.05 143.27 3135056 2540231 258.691 3354.946 227 11929 69.59 90.71 6078868 2181815 6078868 2181815 79.97 86.63 6078868 2507120 6078868 2083806 5597700 3.65 1.38 0 14.13 0 0.37 0 0.30 0 0.00 0 38.63 0 3135056 0 50 0 49.01 0 1.19 0 0.00 0 1.02 0 0.00 0 281.70 0 0.85 0 71265 0 5164546 0 729713 0 18935 0 15717 0 0 0 1994838 0 3 0 0 0 54 0 9845 0 348 0 10250 0 46.57 0 2405343 0 1833 5732 3.127114020731 5164546.0 3135056.0 71265.0 729713.0 18935.0 15717.0 0.0 1994838.0 2405343.0 60.7 1.4 14.1 0.4 0.3 0.0 38.6 46.6 25 25 25.00 7 129113650 27.2 22.4 22.5 27.8 0.0 31.3 17.6 smartseq 876840 SRR2042612 SRP058773 SRS947443 SRX1040889 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697182: q6_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;38 GEO Accession;;GSM1697182 GSM1697182 q6_0 15637950 312759 2015-07-16 16:21:14 9256399 15637950 312759 2 312759 index:0,count:312759,average:25,stdev:0|index:1,count:312759,average:25,stdev:0 GSM1697182_r1 GEO 2.2 2.48 0.49 6655117 7109602 5964698 6492234 106.83 108.84 136592 124100 227.590 5644.134 228 664 36.46 40.83 188218 49807 188218 49807 39.19 39.19 188218 53527 188218 47798 1929925 29.00 1.42 0 4.67 0 0.54 0 1.80 0 0.00 0 53.99 0 136592 0 50 0 49.06 0 1.27 0 0.00 0 1.14 0 0.00 0 43.31 0 0.79 0 4428 0 312759 0 14612 0 1682 0 5633 0 0 0 168852 0 0 0 0 0 1 0 140 0 26 0 167 0 39.00 0 121980 0 32 45 1.406250000000 312759.0 136592.0 4428.0 14612.0 1682.0 5633.0 0.0 168852.0 121980.0 43.7 1.4 4.7 0.5 1.8 0.0 54.0 39.0 25 25 25.00 7 7818975 28.2 17.8 18.1 35.9 0.0 31.3 17.5 smartseq 876857 SRR2042613 SRP058773 SRS947442 SRX1040890 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697183: q7_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;76 GEO Accession;;GSM1697183 GSM1697183 q7_0 109113500 2182270 2015-07-16 16:21:14 64085086 109113500 2182270 2 2182270 index:0,count:2182270,average:25,stdev:0|index:1,count:2182270,average:25,stdev:0 GSM1697183_r1 GEO 1.66 2.48 0.57 60292242 65154505 54333357 60196630 108.06 110.79 1227114 1120205 236.161 3536.335 204 5670 34.65 38.49 1680782 425191 1680782 425191 37.0 36.95 1680782 453989 1680782 408203 20483523 33.97 1.26 0 5.61 0 0.53 0 1.04 0 0.00 0 42.19 0 1227114 0 50 0 49.21 0 1.15 0 0.00 0 1.03 0 0.00 0 191.61 0 0.80 0 27551 0 2182270 0 122486 0 11666 0 22799 0 0 0 920691 0 0 0 0 0 11 0 1411 0 153 0 1575 0 50.62 0 1104628 0 371 565 1.522911051213 2182270.0 1227114.0 27551.0 122486.0 11666.0 22799.0 0.0 920691.0 1104628.0 56.2 1.3 5.6 0.5 1.0 0.0 42.2 50.6 25 25 25.00 7 54556750 29.5 20.5 21.0 29.0 0.0 30.7 16.8 smartseq 876872 SRR2042614 SRP058773 SRS947441 SRX1040891 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697184: q8_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;23 GEO Accession;;GSM1697184 GSM1697184 q8_0 165804500 3316090 2015-07-16 16:21:14 96862680 165804500 3316090 2 3316090 index:0,count:3316090,average:25,stdev:0|index:1,count:3316090,average:25,stdev:0 GSM1697184_r1 GEO 3.77 1.77 0.5 84746791 92468046 71960517 82006841 109.11 113.96 1728173 1560329 238.037 3938.556 227 7604 36.16 42.64 2862103 624896 2862103 624896 40.78 40.98 2862103 704766 2862103 600494 22980729 27.12 1.38 0 7.92 0 0.67 0 1.01 0 0.00 0 46.21 0 1728173 0 50 0 49.18 0 1.23 0 0.00 0 1.03 0 0.00 0 397.93 0 0.81 0 45687 0 3316090 0 262760 0 22161 0 33467 0 0 0 1532289 0 2 0 0 0 27 0 2397 0 247 0 2673 0 44.19 0 1465413 0 614 1129 1.838762214984 3316090.0 1728173.0 45687.0 262760.0 22161.0 33467.0 0.0 1532289.0 1465413.0 52.1 1.4 7.9 0.7 1.0 0.0 46.2 44.2 25 25 25.00 7 82902250 28.4 21.6 21.4 28.6 0.0 31.0 17.3 smartseq 876920 SRR2042617 SRP058773 SRS947438 SRX1040894 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697187: q11_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;40 GEO Accession;;GSM1697187 GSM1697187 q11_0 74284650 1485693 2015-07-16 16:21:14 43564365 74284650 1485693 2 1485693 index:0,count:1485693,average:25,stdev:0|index:1,count:1485693,average:25,stdev:0 GSM1697187_r1 GEO 1.22 1.31 2.23 34322891 23822311 24782000 19318241 69.41 77.95 703386 618965 229.606 6839.488 218 3218 15.12 21.0 1692362 106332 1692362 106332 20.34 20.06 1692362 143044 1692362 101574 8494452 24.75 1.81 0 13.26 0 2.75 0 2.04 0 0.00 0 47.87 0 703386 0 50 0 49.05 0 1.21 0 0.00 0 1.01 0 0.00 0 148.57 0 0.84 0 26935 0 1485693 0 197066 0 40794 0 30359 0 0 0 711154 0 0 0 0 0 8 0 535 0 131 0 674 0 34.08 0 506320 0 163 229 1.404907975460 1485693.0 703386.0 26935.0 197066.0 40794.0 30359.0 0.0 711154.0 506320.0 47.3 1.8 13.3 2.7 2.0 0.0 47.9 34.1 25 25 25.00 7 37142325 27.9 21.2 21.1 29.8 0.0 31.2 17.6 smartseq 876936 SRR2042618 SRP058773 SRS947437 SRX1040895 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697188: q12_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;14 GEO Accession;;GSM1697188 GSM1697188 q12_0 230144750 4602895 2015-07-16 16:21:14 134080874 230144750 4602895 2 4602895 index:0,count:4602895,average:25,stdev:0|index:1,count:4602895,average:25,stdev:0 GSM1697188_r1 GEO 2.95 2.26 0.38 125479783 142171955 108309088 126664741 113.3 116.95 2558934 2271741 239.424 3804.285 217 11086 42.88 49.74 3910404 1097391 3910404 1097391 47.36 47.56 3910404 1211870 3910404 1049312 30438140 24.26 1.37 0 7.66 0 0.63 0 0.94 0 0.00 0 42.84 0 2558934 0 50 0 49.15 0 1.16 0 0.00 0 1.01 0 0.00 0 91.05 0 0.80 0 63107 0 4602895 0 352542 0 28984 0 43141 0 0 0 1971836 0 3 0 0 0 36 0 4045 0 395 0 4479 0 47.93 0 2206392 0 956 1936 2.025104602510 4602895.0 2558934.0 63107.0 352542.0 28984.0 43141.0 0.0 1971836.0 2206392.0 55.6 1.4 7.7 0.6 0.9 0.0 42.8 47.9 25 25 25.00 7 115072375 28.5 21.4 21.2 28.9 0.0 31.2 17.5 smartseq 876953 SRR2042619 SRP058773 SRS947436 SRX1040896 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697189: q13_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;35 GEO Accession;;GSM1697189 GSM1697189 q13_0 185549500 3710990 2015-07-16 16:21:14 108063426 185549500 3710990 2 3710990 index:0,count:3710990,average:25,stdev:0|index:1,count:3710990,average:25,stdev:0 GSM1697189_r1 GEO 3.88 1.27 1.06 91820331 87377447 69248380 71888993 95.16 103.81 1879822 1657658 233.972 5536.757 207 8356 32.59 43.29 4130580 612626 4130580 612626 39.96 41.51 4130580 751169 4130580 587493 18287483 19.92 1.72 0 12.52 0 1.31 0 1.37 0 0.00 0 46.66 0 1879822 0 50 0 49.01 0 1.20 0 0.00 0 1.01 0 0.00 0 261.95 0 0.82 0 63968 0 3710990 0 464570 0 48517 0 50927 0 0 0 1731724 0 0 0 0 0 33 0 2751 0 343 0 3127 0 38.14 0 1415252 0 597 1322 2.214405360134 3710990.0 1879822.0 63968.0 464570.0 48517.0 50927.0 0.0 1731724.0 1415252.0 50.7 1.7 12.5 1.3 1.4 0.0 46.7 38.1 25 25 25.00 7 92774750 27.5 21.7 21.7 29.1 0.0 31.2 17.5 smartseq 877064 SRR2042620 SRP058773 SRS947434 SRX1040897 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697190: q14_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;56 GEO Accession;;GSM1697190 GSM1697190 q14_0 85072650 1701453 2015-07-16 16:21:14 49841589 85072650 1701453 2 1701453 index:0,count:1701453,average:25,stdev:0|index:1,count:1701453,average:25,stdev:0 GSM1697190_r1 GEO 5.01 1.97 0.22 44848962 57689302 33358120 47149302 128.63 141.34 917374 746599 246.261 4166.438 207 3742 68.78 92.5 1953567 631002 1953567 631002 79.17 88.23 1953567 726301 1953567 601847 1268755 2.83 1.30 0 13.82 0 0.42 0 0.80 0 0.00 0 44.86 0 917374 0 50 0 48.98 0 1.30 0 0.00 0 1.01 0 0.00 0 161.19 0 0.87 0 22112 0 1701453 0 235211 0 7206 0 13620 0 0 0 763253 0 0 0 0 0 18 0 2820 0 106 0 2944 0 40.09 0 682163 0 585 1345 2.299145299145 1701453.0 917374.0 22112.0 235211.0 7206.0 13620.0 0.0 763253.0 682163.0 53.9 1.3 13.8 0.4 0.8 0.0 44.9 40.1 25 25 25.00 7 42536325 26.7 21.7 21.4 30.1 0.0 31.2 17.6 smartseq 877080 SRR2042621 SRP058773 SRS947435 SRX1040898 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697191: q15_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;39 GEO Accession;;GSM1697191 GSM1697191 q15_0 106349400 2126988 2015-07-16 16:21:14 62352657 106349400 2126988 2 2126988 index:0,count:2126988,average:25,stdev:0|index:1,count:2126988,average:25,stdev:0 GSM1697191_r1 GEO 1.84 2.69 0.83 56554802 60779989 51132955 56700203 107.47 110.89 1152322 1057119 233.814 3980.944 219 5189 31.75 35.18 1585827 365876 1585827 365876 33.64 33.84 1585827 387655 1585827 351939 20960516 37.06 1.33 0 5.27 0 0.83 0 1.11 0 0.00 0 43.89 0 1152322 0 50 0 49.24 0 1.24 0 0.00 0 1.02 0 0.00 0 182.31 0 0.79 0 28271 0 2126988 0 112193 0 17671 0 23545 0 0 0 933450 0 1 0 0 0 18 0 1246 0 145 0 1410 0 48.90 0 1040129 0 273 428 1.567765567766 2126988.0 1152322.0 28271.0 112193.0 17671.0 23545.0 0.0 933450.0 1040129.0 54.2 1.3 5.3 0.8 1.1 0.0 43.9 48.9 25 25 25.00 7 53174700 29.4 20.2 20.3 30.1 0.0 31.1 17.5 smartseq 877097 SRR2042622 SRP058773 SRS947433 SRX1040899 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697192: q16_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;18 GEO Accession;;GSM1697192 GSM1697192 q16_0 78027300 1560546 2015-07-16 16:21:14 45520489 78027300 1560546 2 1560546 index:0,count:1560546,average:25,stdev:0|index:1,count:1560546,average:25,stdev:0 GSM1697192_r1 GEO 1.46 1.49 1.83 32955405 24592648 25597866 20653701 74.62 80.69 677269 596915 215.539 7765.094 212 3075 16.83 21.74 1388640 114004 1388640 114004 21.51 20.91 1388640 145703 1388640 109688 9359977 28.40 1.94 0 9.79 0 1.72 0 2.02 0 0.00 0 52.86 0 677269 0 50 0 48.94 0 1.15 0 0.00 0 1.00 0 0.00 0 175.56 0 0.82 0 30347 0 1560546 0 152786 0 26831 0 31582 0 0 0 824864 0 1 0 0 0 9 0 658 0 168 0 836 0 33.61 0 524483 0 140 230 1.642857142857 1560546.0 677269.0 30347.0 152786.0 26831.0 31582.0 0.0 824864.0 524483.0 43.4 1.9 9.8 1.7 2.0 0.0 52.9 33.6 25 25 25.00 7 39013650 27.6 20.5 20.3 31.5 0.0 31.3 17.6 smartseq 877160 SRR2042626 SRP058773 SRS947428 SRX1040903 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697196: q20_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;59 GEO Accession;;GSM1697196 GSM1697196 q20_0 115262050 2305241 2015-07-16 16:21:14 67474541 115262050 2305241 2 2305241 index:0,count:2305241,average:25,stdev:0|index:1,count:2305241,average:25,stdev:0 GSM1697196_r1 GEO 6.12 1.77 0.17 60181859 78919419 44724266 64150027 131.13 143.43 1230915 1017912 238.820 3768.912 207 5105 69.07 92.95 2592744 850253 2592744 850253 80.24 89.17 2592744 987744 2592744 815636 1396811 2.32 1.28 0 13.72 0 0.38 0 0.54 0 0.00 0 45.69 0 1230915 0 50 0 48.98 0 1.23 0 0.00 0 1.00 0 0.00 0 169.36 0 0.89 0 29432 0 2305241 0 316196 0 8806 0 12345 0 0 0 1053175 0 1 0 0 0 31 0 3599 0 149 0 3780 0 39.68 0 914719 0 849 1857 2.187279151943 2305241.0 1230915.0 29432.0 316196.0 8806.0 12345.0 0.0 1053175.0 914719.0 53.4 1.3 13.7 0.4 0.5 0.0 45.7 39.7 25 25 25.00 7 57631025 27.3 21.9 21.6 29.2 0.0 31.0 17.4 smartseq 877176 SRR2042627 SRP058773 SRS947429 SRX1040904 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697197: q21_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;89 GEO Accession;;GSM1697197 GSM1697197 q21_0 80351500 1607030 2015-07-16 16:21:14 46888365 80351500 1607030 2 1607030 index:0,count:1607030,average:25,stdev:0|index:1,count:1607030,average:25,stdev:0 GSM1697197_r1 GEO 1.28 2.02 0.8 38527270 35538948 33297328 31610919 92.24 94.94 785398 697511 237.918 5746.763 218 3308 30.95 35.89 1199509 243089 1199509 243089 34.22 34.12 1199509 268750 1199509 231111 10258958 26.63 1.68 0 6.73 0 0.65 0 2.78 0 0.00 0 47.69 0 785398 0 50 0 49.26 0 1.23 0 0.00 0 1.00 0 0.00 0 180.79 0 0.75 0 27016 0 1607030 0 108117 0 10511 0 44714 0 0 0 766407 0 3 0 0 0 16 0 1037 0 139 0 1195 0 42.14 0 677281 0 216 517 2.393518518519 1607030.0 785398.0 27016.0 108117.0 10511.0 44714.0 0.0 766407.0 677281.0 48.9 1.7 6.7 0.7 2.8 0.0 47.7 42.1 25 25 25.00 7 40175750 26.3 20.8 20.4 32.4 0.0 31.3 17.5 smartseq 877192 SRR2042628 SRP058773 SRS947427 SRX1040905 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697198: q22_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;74 GEO Accession;;GSM1697198 GSM1697198 q22_0 85895750 1717915 2015-07-16 16:21:14 50229314 85895750 1717915 2 1717915 index:0,count:1717915,average:25,stdev:0|index:1,count:1717915,average:25,stdev:0 GSM1697198_r1 GEO 4.51 2.18 0.36 46090534 55948278 40358430 50360007 121.39 124.78 939901 828659 253.289 3873.094 227 3834 48.7 55.69 1374466 457694 1374466 457694 53.14 53.68 1374466 499498 1374466 441199 11035971 23.94 1.24 0 6.87 0 0.45 0 1.16 0 0.00 0 43.68 0 939901 0 50 0 49.17 0 1.18 0 0.00 0 1.06 0 0.00 0 206.15 0 0.78 0 21241 0 1717915 0 117973 0 7717 0 19997 0 0 0 750300 0 0 0 0 0 9 0 1724 0 118 0 1851 0 47.84 0 821928 0 366 818 2.234972677596 1717915.0 939901.0 21241.0 117973.0 7717.0 19997.0 0.0 750300.0 821928.0 54.7 1.2 6.9 0.4 1.2 0.0 43.7 47.8 25 25 25.00 7 42947875 27.8 20.3 20.4 31.5 0.0 31.3 17.7 smartseq 877208 SRR2042629 SRP058773 SRS947426 SRX1040906 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697199: q23_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;92 GEO Accession;;GSM1697199 GSM1697199 q23_0 132776850 2655537 2015-07-16 16:21:14 77589523 132776850 2655537 2 2655537 index:0,count:2655537,average:25,stdev:0|index:1,count:2655537,average:25,stdev:0 GSM1697199_r1 GEO 7.93 1.48 0.2 70543843 93988801 51968868 76499341 133.23 147.2 1444173 1193398 248.611 3644.636 207 5677 68.56 93.06 3115237 990064 3115237 990064 79.55 89.03 3115237 1148793 3115237 947189 1668049 2.36 1.29 0 14.32 0 0.46 0 0.65 0 0.00 0 44.50 0 1444173 0 50 0 48.94 0 1.20 0 0.00 0 1.01 0 0.00 0 187.45 0 0.89 0 34232 0 2655537 0 380287 0 12287 0 17243 0 0 0 1181834 0 1 0 0 0 28 0 4345 0 187 0 4561 0 40.06 0 1063886 0 905 2399 2.650828729282 2655537.0 1444173.0 34232.0 380287.0 12287.0 17243.0 0.0 1181834.0 1063886.0 54.4 1.3 14.3 0.5 0.6 0.0 44.5 40.1 25 25 25.00 7 66388425 26.5 22.5 22.3 28.6 0.0 31.1 17.3 smartseq 877320 SRR2042630 SRP058773 SRS947425 SRX1040907 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697200: q24_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;21 GEO Accession;;GSM1697200 GSM1697200 q24_0 146565200 2931304 2015-07-16 16:21:14 85586163 146565200 2931304 2 2931304 index:0,count:2931304,average:25,stdev:0|index:1,count:2931304,average:25,stdev:0 GSM1697200_r1 GEO 2.48 2.7 0.37 80171531 86691546 71297509 78789203 108.13 110.51 1634050 1485458 235.684 3701.911 218 7237 35.06 39.47 2313056 572841 2313056 572841 38.17 37.83 2313056 623642 2313056 549054 24720156 30.83 1.36 0 6.23 0 0.63 0 1.19 0 0.00 0 42.43 0 1634050 0 50 0 49.20 0 1.24 0 0.00 0 1.02 0 0.00 0 234.50 0 0.76 0 39731 0 2931304 0 182665 0 18450 0 34907 0 0 0 1243897 0 3 0 0 0 17 0 2025 0 252 0 2297 0 49.51 0 1451385 0 492 915 1.859756097561 2931304.0 1634050.0 39731.0 182665.0 18450.0 34907.0 0.0 1243897.0 1451385.0 55.7 1.4 6.2 0.6 1.2 0.0 42.4 49.5 25 25 25.00 7 73282600 28.8 20.8 20.7 29.7 0.0 31.2 17.6 smartseq 877338 SRR2042631 SRP058773 SRS947423 SRX1040908 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697201: q25_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;63 GEO Accession;;GSM1697201 GSM1697201 q25_0 101030800 2020616 2015-07-16 16:21:14 59208616 101030800 2020616 2 2020616 index:0,count:2020616,average:25,stdev:0|index:1,count:2020616,average:25,stdev:0 GSM1697201_r1 GEO 8.52 2.01 0.19 47588418 63391930 35830383 51990657 133.21 145.1 975589 798756 258.839 4373.531 227 3742 68.22 90.65 1993180 665559 1993180 665559 78.92 86.87 1993180 769930 1993180 637753 1752745 3.68 1.27 0 11.95 0 0.39 0 0.58 0 0.00 0 50.75 0 975589 0 50 0 48.88 0 1.16 0 0.00 0 1.01 0 0.00 0 134.71 0 0.91 0 25710 0 2020616 0 241406 0 7902 0 11662 0 0 0 1025463 0 3 0 0 0 22 0 2847 0 134 0 3006 0 36.33 0 734183 0 567 1442 2.543209876543 2020616.0 975589.0 25710.0 241406.0 7902.0 11662.0 0.0 1025463.0 734183.0 48.3 1.3 11.9 0.4 0.6 0.0 50.8 36.3 25 25 25.00 7 50515400 27.3 21.7 21.9 29.1 0.0 31.0 17.2 smartseq 877354 SRR2042632 SRP058773 SRS947424 SRX1040909 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697202: q26_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;58 GEO Accession;;GSM1697202 GSM1697202 q26_0 203830600 4076612 2015-07-16 16:21:14 118997966 203830600 4076612 2 4076612 index:0,count:4076612,average:25,stdev:0|index:1,count:4076612,average:25,stdev:0 GSM1697202_r1 GEO 2.02 2.98 0.36 111948250 122668156 102974882 114557667 109.58 111.25 2276937 2109503 232.988 3673.912 218 10629 32.25 35.1 2897234 734272 2897234 734272 33.98 33.84 2897234 773691 2897234 708012 38768037 34.63 1.34 0 4.54 0 0.48 0 1.05 0 0.00 0 42.61 0 2276937 0 50 0 49.28 0 1.19 0 0.00 0 1.03 0 0.00 0 266.83 0 0.76 0 54779 0 4076612 0 185012 0 19604 0 42870 0 0 0 1737201 0 2 0 0 0 25 0 2257 0 347 0 2631 0 51.32 0 2091925 0 474 897 1.892405063291 4076612.0 2276937.0 54779.0 185012.0 19604.0 42870.0 0.0 1737201.0 2091925.0 55.9 1.3 4.5 0.5 1.1 0.0 42.6 51.3 25 25 25.00 7 101915300 29.0 20.7 20.2 30.0 0.0 31.2 17.8 smartseq 877370 SRR2042633 SRP058773 SRS947422 SRX1040910 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697203: q27_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;84 GEO Accession;;GSM1697203 GSM1697203 q27_0 183912700 3678254 2015-07-16 16:21:14 107159216 183912700 3678254 2 3678254 index:0,count:3678254,average:25,stdev:0|index:1,count:3678254,average:25,stdev:0 GSM1697203_r1 GEO 1.77 2.77 0.28 102382636 114743188 89945396 103539896 112.07 115.11 2086989 1871796 239.211 3846.695 217 9060 39.43 44.94 3081209 822953 3081209 822953 43.31 43.04 3081209 903936 3081209 788237 28981321 28.31 1.32 0 6.95 0 0.44 0 1.04 0 0.00 0 41.77 0 2086989 0 50 0 49.19 0 1.17 0 0.00 0 1.01 0 0.00 0 213.58 0 0.75 0 48540 0 3678254 0 255741 0 16339 0 38385 0 0 0 1536541 0 4 0 0 0 37 0 3124 0 287 0 3452 0 49.79 0 1831248 0 639 1600 2.503912363067 3678254.0 2086989.0 48540.0 255741.0 16339.0 38385.0 0.0 1536541.0 1831248.0 56.7 1.3 7.0 0.4 1.0 0.0 41.8 49.8 25 25 25.00 7 91956350 28.4 20.6 20.7 30.3 0.0 31.2 17.6 smartseq 877387 SRR2042634 SRP058773 SRS947420 SRX1040911 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697204: q28_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;16 GEO Accession;;GSM1697204 GSM1697204 q28_0 44146750 882935 2015-07-16 16:21:14 25861794 44146750 882935 2 882935 index:0,count:882935,average:25,stdev:0|index:1,count:882935,average:25,stdev:0 GSM1697204_r1 GEO 8.14 2.04 0.22 19771484 26233001 14664933 21222340 132.68 144.71 406096 330674 244.005 5562.557 207 1642 67.98 91.77 849024 276073 849024 276073 79.23 87.76 849024 321767 849024 264000 667737 3.38 1.25 0 11.92 0 0.48 0 1.16 0 0.00 0 52.36 0 406096 0 50 0 48.85 0 1.33 0 0.00 0 1.00 0 0.00 0 122.25 0 0.91 0 11052 0 882935 0 105276 0 4246 0 10281 0 0 0 462312 0 0 0 0 0 3 0 1235 0 65 0 1303 0 34.07 0 300820 0 294 582 1.979591836735 882935.0 406096.0 11052.0 105276.0 4246.0 10281.0 0.0 462312.0 300820.0 46.0 1.3 11.9 0.5 1.2 0.0 52.4 34.1 25 25 25.00 7 22073375 26.0 20.3 20.3 33.3 0.0 31.3 17.5 smartseq 877403 SRR2042635 SRP058773 SRS947421 SRX1040912 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697205: q29_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;88 GEO Accession;;GSM1697205 GSM1697205 q29_0 296214100 5924282 2015-07-16 16:21:14 172472495 296214100 5924282 2 5924282 index:0,count:5924282,average:25,stdev:0|index:1,count:5924282,average:25,stdev:0 GSM1697205_r1 GEO 5.18 1.86 0.22 166314061 211026275 131316391 177544336 126.88 135.2 3398937 2807808 250.319 3501.395 207 13286 62.7 79.43 6379131 2131190 6379131 2131190 71.46 75.94 6379131 2428741 6379131 2037549 14215608 8.55 1.30 0 12.08 0 0.40 0 0.54 0 0.00 0 41.69 0 3398937 0 50 0 49.01 0 1.20 0 0.00 0 1.01 0 0.00 0 239.63 0 0.83 0 76770 0 5924282 0 715790 0 23452 0 31822 0 0 0 2470071 0 9 0 0 0 60 0 9189 0 440 0 9698 0 45.29 0 2683147 0 1724 5172 3.000000000000 5924282.0 3398937.0 76770.0 715790.0 23452.0 31822.0 0.0 2470071.0 2683147.0 57.4 1.3 12.1 0.4 0.5 0.0 41.7 45.3 25 25 25.00 7 148107050 27.5 22.3 22.1 28.0 0.0 31.1 17.4 smartseq 877420 SRR2042636 SRP058773 SRS947419 SRX1040913 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697206: q30_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;54 GEO Accession;;GSM1697206 GSM1697206 q30_0 52793350 1055867 2015-07-16 16:21:14 30893049 52793350 1055867 2 1055867 index:0,count:1055867,average:25,stdev:0|index:1,count:1055867,average:25,stdev:0 GSM1697206_r1 GEO 6.34 1.73 0.21 22839242 30374130 17538226 25286776 132.99 144.18 468128 382686 240.385 5033.002 207 1932 71.75 93.54 936066 335877 936066 335877 81.09 89.77 936066 379624 936066 322338 572413 2.51 1.20 0 10.33 0 0.26 0 1.73 0 0.00 0 53.67 0 468128 0 50 0 48.94 0 1.21 0 0.00 0 1.03 0 0.00 0 146.20 0 0.88 0 12667 0 1055867 0 109060 0 2757 0 18260 0 0 0 566722 0 3 0 0 0 15 0 1313 0 64 0 1395 0 34.01 0 359068 0 294 609 2.071428571429 1055867.0 468128.0 12667.0 109060.0 2757.0 18260.0 0.0 566722.0 359068.0 44.3 1.2 10.3 0.3 1.7 0.0 53.7 34.0 25 25 25.00 7 26396675 26.3 20.0 19.8 34.0 0.0 31.3 17.7 smartseq 877435 SRR2042637 SRP058773 SRS947418 SRX1040914 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697207: q31_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;45 GEO Accession;;GSM1697207 GSM1697207 q31_0 121196650 2423933 2015-07-16 16:21:14 70951568 121196650 2423933 2 2423933 index:0,count:2423933,average:25,stdev:0|index:1,count:2423933,average:25,stdev:0 GSM1697207_r1 GEO 1.94 2.63 0.78 65568238 66080518 59009177 60844135 100.78 103.11 1336389 1233051 226.617 3965.344 217 6120 28.58 31.8 1829585 381880 1829585 381880 30.98 30.54 1829585 414001 1829585 366796 22335248 34.06 1.39 0 5.59 0 0.78 0 1.29 0 0.00 0 42.80 0 1336389 0 50 0 49.23 0 1.16 0 0.00 0 1.02 0 0.00 0 249.32 0 0.76 0 33762 0 2423933 0 135529 0 18893 0 31171 0 0 0 1037480 0 0 0 0 0 14 0 1362 0 205 0 1581 0 49.54 0 1200860 0 360 581 1.613888888889 2423933.0 1336389.0 33762.0 135529.0 18893.0 31171.0 0.0 1037480.0 1200860.0 55.1 1.4 5.6 0.8 1.3 0.0 42.8 49.5 25 25 25.00 7 60598325 29.0 20.4 20.5 30.2 0.0 31.2 17.7 smartseq 877451 SRR2042638 SRP058773 SRS947417 SRX1040915 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697208: q32_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;26 GEO Accession;;GSM1697208 GSM1697208 q32_0 234286300 4685726 2015-07-16 16:21:14 136465539 234286300 4685726 2 4685726 index:0,count:4685726,average:25,stdev:0|index:1,count:4685726,average:25,stdev:0 GSM1697208_r1 GEO 5.31 1.9 0.21 135906057 168611336 111372053 144959770 124.06 130.16 2771022 2337576 266.110 3422.812 240 10644 56.59 69.09 4787302 1568172 4787302 1568172 63.96 66.18 4787302 1772266 4787302 1501971 20617045 15.17 1.35 0 10.70 0 0.49 0 0.54 0 0.00 0 39.83 0 2771022 0 50 0 49.11 0 1.21 0 0.00 0 1.01 0 0.00 0 272.07 0 0.81 0 63409 0 4685726 0 501373 0 22926 0 25233 0 0 0 1866545 0 2 0 0 0 33 0 6594 0 351 0 6980 0 48.44 0 2269649 0 1488 3386 2.275537634409 4685726.0 2771022.0 63409.0 501373.0 22926.0 25233.0 0.0 1866545.0 2269649.0 59.1 1.4 10.7 0.5 0.5 0.0 39.8 48.4 25 25 25.00 7 117143150 27.8 21.9 21.8 28.5 0.0 31.3 17.6 smartseq 877466 SRR2042639 SRP058773 SRS947416 SRX1040916 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697209: q33_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;68 GEO Accession;;GSM1697209 GSM1697209 q33_0 235585100 4711702 2015-07-16 16:21:14 137545451 235585100 4711702 2 4711702 index:0,count:4711702,average:25,stdev:0|index:1,count:4711702,average:25,stdev:0 GSM1697209_r1 GEO 4.31 2.12 0.32 130133602 154001476 112466580 137477164 118.34 122.24 2655961 2338341 256.409 3561.805 227 10730 44.04 51.0 4084544 1169564 4084544 1169564 49.0 49.11 4084544 1301324 4084544 1126274 35066450 26.95 1.30 0 7.70 0 0.47 0 0.70 0 0.00 0 42.46 0 2655961 0 50 0 49.10 0 1.18 0 0.00 0 1.01 0 0.00 0 257.00 0 0.80 0 61107 0 4711702 0 362665 0 22117 0 33128 0 0 0 2000496 0 14 0 0 0 37 0 4887 0 346 0 5284 0 48.67 0 2293296 0 1003 2496 2.488534396810 4711702.0 2655961.0 61107.0 362665.0 22117.0 33128.0 0.0 2000496.0 2293296.0 56.4 1.3 7.7 0.5 0.7 0.0 42.5 48.7 25 25 25.00 7 117792550 28.5 21.4 21.5 28.6 0.0 31.0 17.3 smartseq 877578 SRR2042640 SRP058773 SRS947405 SRX1040917 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697210: q34_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;72 GEO Accession;;GSM1697210 GSM1697210 q34_0 172272550 3445451 2015-07-16 16:21:14 100597796 172272550 3445451 2 3445451 index:0,count:3445451,average:25,stdev:0|index:1,count:3445451,average:25,stdev:0 GSM1697210_r1 GEO 4.48 1.12 0.58 84780132 93034530 57993924 73071014 109.74 126.0 1739206 1483895 242.627 4755.864 207 7392 47.5 69.47 4534402 826165 4534402 826165 58.48 66.58 4534402 1017053 4534402 791708 8016077 9.46 1.53 0 15.96 0 1.01 0 0.80 0 0.00 0 47.71 0 1739206 0 50 0 48.85 0 1.19 0 0.00 0 1.02 0 0.00 0 167.62 0 0.87 0 52800 0 3445451 0 550020 0 34871 0 27698 0 0 0 1643676 0 1 0 0 0 29 0 3659 0 279 0 3968 0 34.51 0 1189186 0 761 1774 2.331143232589 3445451.0 1739206.0 52800.0 550020.0 34871.0 27698.0 0.0 1643676.0 1189186.0 50.5 1.5 16.0 1.0 0.8 0.0 47.7 34.5 25 25 25.00 7 86136275 27.2 22.4 22.6 27.9 0.0 31.1 17.3 smartseq 877594 SRR2042641 SRP058773 SRS947406 SRX1040918 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697211: q35_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;11 GEO Accession;;GSM1697211 GSM1697211 q35_0 72666500 1453330 2015-07-16 16:21:14 42563447 72666500 1453330 2 1453330 index:0,count:1453330,average:25,stdev:0|index:1,count:1453330,average:25,stdev:0 GSM1697211_r1 GEO 2.49 2.56 0.33 36184240 39294689 31957025 35534100 108.6 111.19 739438 675614 215.468 5061.494 197 3546 34.07 38.66 1066182 251913 1066182 251913 37.25 37.06 1066182 275419 1066182 241495 11358268 31.39 1.57 0 6.04 0 0.43 0 1.52 0 0.00 0 47.17 0 739438 0 50 0 49.13 0 1.22 0 0.00 0 1.02 0 0.00 0 58.79 0 0.73 0 22887 0 1453330 0 87765 0 6262 0 22161 0 0 0 685469 0 0 0 0 0 6 0 901 0 158 0 1065 0 44.84 0 651673 0 219 340 1.552511415525 1453330.0 739438.0 22887.0 87765.0 6262.0 22161.0 0.0 685469.0 651673.0 50.9 1.6 6.0 0.4 1.5 0.0 47.2 44.8 25 25 25.00 7 36333250 28.3 19.7 19.9 32.1 0.0 31.1 17.3 smartseq 877610 SRR2042642 SRP058773 SRS947404 SRX1040919 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697212: q36_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;30 GEO Accession;;GSM1697212 GSM1697212 q36_0 268839700 5376794 2015-07-16 16:21:14 156364127 268839700 5376794 2 5376794 index:0,count:5376794,average:25,stdev:0|index:1,count:5376794,average:25,stdev:0 GSM1697212_r1 GEO 5.02 1.72 0.24 154566674 189888771 121176774 159289067 122.85 131.45 3154171 2664429 253.128 3411.201 207 12679 56.99 72.71 6124813 1797430 6124813 1797430 65.76 69.52 6124813 2074203 6124813 1718471 18492911 11.96 1.38 0 12.69 0 0.44 0 0.51 0 0.00 0 40.39 0 3154171 0 50 0 49.08 0 1.15 0 0.00 0 1.01 0 0.00 0 201.63 0 0.81 0 74458 0 5376794 0 682256 0 23497 0 27308 0 0 0 2171818 0 4 0 0 0 63 0 7596 0 344 0 8007 0 45.97 0 2471915 0 1481 4219 2.848750844024 5376794.0 3154171.0 74458.0 682256.0 23497.0 27308.0 0.0 2171818.0 2471915.0 58.7 1.4 12.7 0.4 0.5 0.0 40.4 46.0 25 25 25.00 7 134419850 27.9 21.7 21.8 28.7 0.0 31.2 17.6 smartseq 877626 SRR2042643 SRP058773 SRS947403 SRX1040920 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697213: q37_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;65 GEO Accession;;GSM1697213 GSM1697213 q37_0 63062750 1261255 2015-07-16 16:21:14 36927441 63062750 1261255 2 1261255 index:0,count:1261255,average:25,stdev:0|index:1,count:1261255,average:25,stdev:0 GSM1697213_r1 GEO 6.63 1.59 0.19 31182106 41282902 23169394 33911131 132.39 146.36 640629 514496 258.418 5149.459 227 2463 69.58 93.71 1372257 445722 1372257 445722 79.35 89.22 1372257 508338 1372257 424331 762027 2.44 1.25 0 13.08 0 0.39 0 1.07 0 0.00 0 47.75 0 640629 0 50 0 48.82 0 1.31 0 0.00 0 1.07 0 0.00 0 162.16 0 0.88 0 15803 0 1261255 0 165012 0 4961 0 13472 0 0 0 602193 0 6 0 0 0 15 0 2046 0 108 0 2175 0 37.71 0 475617 0 372 987 2.653225806452 1261255.0 640629.0 15803.0 165012.0 4961.0 13472.0 0.0 602193.0 475617.0 50.8 1.3 13.1 0.4 1.1 0.0 47.7 37.7 25 25 25.00 7 31531375 26.0 21.1 21.2 31.7 0.0 31.2 17.4 smartseq 877642 SRR2042644 SRP058773 SRS947402 SRX1040921 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697214: q38_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;44 GEO Accession;;GSM1697214 GSM1697214 q38_0 150336450 3006729 2015-07-16 16:21:14 87959590 150336450 3006729 2 3006729 index:0,count:3006729,average:25,stdev:0|index:1,count:3006729,average:25,stdev:0 GSM1697214_r1 GEO 2.56 2.86 0.54 81784091 91125695 72711173 83412340 111.42 114.72 1666118 1519663 234.744 3722.921 218 7408 35.46 39.95 2401107 590868 2401107 590868 38.51 38.41 2401107 641645 2401107 568106 26929483 32.93 1.34 0 6.22 0 0.65 0 0.97 0 0.00 0 42.96 0 1666118 0 50 0 49.23 0 1.22 0 0.00 0 1.02 0 0.00 0 240.54 0 0.79 0 40147 0 3006729 0 186942 0 19582 0 29222 0 0 0 1291807 0 2 0 0 0 14 0 1996 0 219 0 2231 0 49.20 0 1479176 0 525 902 1.718095238095 3006729.0 1666118.0 40147.0 186942.0 19582.0 29222.0 0.0 1291807.0 1479176.0 55.4 1.3 6.2 0.7 1.0 0.0 43.0 49.2 25 25 25.00 7 75168225 28.9 20.7 20.8 29.6 0.0 31.1 17.5 smartseq 877656 SRR2042645 SRP058773 SRS947401 SRX1040922 SRA270094 GEO Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2] Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. Overall design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming GSM1697215: q39_0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired The single-cell RNA-seq libraries were prepared from MEF-depleted hiF-T cells at day 10 of reprogramming directly sorted in lysis buffer at single cell using an ARIA facs sorter (BD). Library preparation was performed using SMART-seq2 protocol with minor modifications (Trombetta et al., 2014) on 96 cells Illumina HiSeq 2500 source_name;;MEF-depleted hiF-T cells at day 10 of reprogramming|well number;;27 GEO Accession;;GSM1697215 GSM1697215 q39_0 69893050 1397861 2015-07-16 16:21:14 40904974 69893050 1397861 2 1397861 index:0,count:1397861,average:25,stdev:0|index:1,count:1397861,average:25,stdev:0 GSM1697215_r1 GEO 3.07 1.92 0.18 34852095 43055609 26046601 35379442 123.54 135.83 713307 588019 253.991 4697.124 207 2871 62.89 84.2 1531932 448570 1531932 448570 72.97 80.71 1531932 520465 1531932 429971 2314084 6.64 1.32 0 12.92 0 0.42 0 0.88 0 0.00 0 47.67 0 713307 0 50 0 48.98 0 1.12 0 0.00 0 1.00 0 0.00 0 39.94 0 0.85 0 18452 0 1397861 0 180576 0 5899 0 12284 0 0 0 666371 0 0 0 0 0 13 0 1985 0 113 0 2111 0 38.11 0 532731 0 431 975 2.262180974478 1397861.0 713307.0 18452.0 180576.0 5899.0 12284.0 0.0 666371.0 532731.0 51.0 1.3 12.9 0.4 0.9 0.0 47.7 38.1 25 25 25.00 7 34946525 26.6 20.9 21.0 31.5 0.0 31.3 17.5 smartseq 124587 SRR2049355 SRP059035 SRS951808 SRX1047435 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702603: H358_SC38; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702603 GSM1702603 H358_SC38 1212826400 6064132 2015-06-05 16:18:03 795233858 1212826400 6064132 2 6064132 index:0,count:6064132,average:100,stdev:0|index:1,count:6064132,average:100,stdev:0 GSM1702603_r1 3.19 3.42 0.12 969850568 1216831950 935616949 1178733931 125.47 125.98 5504001 4856823 264.422 1231.966 165 22323 63.48 65.94 5873493 3493862 5873493 3493862 62.48 62.55 5873493 3438652 5873493 3314590 190907696 19.68 1.38 0 3.38 0 0.14 0 0.06 0 0.00 0 9.04 0 5504001 0 200 0 196.90 0 1.54 0 0.02 0 1.49 0 0.01 0 191.50 0 0.34 0 83876 0 6064132 0 205103 0 8580 0 3451 0 0 0 548100 0 1412 0 0 0 13532 0 1609250 0 12090 0 1636284 0 87.38 0 5298898 0 45070 1584370 35.153538939428 6064132.0 5504001.0 83876.0 205103.0 8580.0 3451.0 0.0 548100.0 5298898.0 90.8 1.4 3.4 0.1 0.1 0.0 9.0 87.4 100 100 100.00 38 606413200 27.9 21.7 21.3 29.2 0.0 35.9 18.9 smartseq 124589 SRR2049356 SRP059035 SRS951807 SRX1047436 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702604: H358_SC39; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702604 GSM1702604 H358_SC39 773628800 3868144 2015-06-05 16:18:03 512090841 773628800 3868144 2 3868144 index:0,count:3868144,average:100,stdev:0|index:1,count:3868144,average:100,stdev:0 GSM1702604_r1 2.95 4.43 0.13 664621216 842045200 640559436 815272000 126.7 127.27 3584899 3015044 310.965 1644.062 183 11094 66.0 68.59 3819392 2366100 3819392 2366100 65.1 65.17 3819392 2333899 3819392 2248283 118360709 17.81 1.30 0 3.49 0 0.17 0 0.08 0 0.00 0 7.07 0 3584899 0 200 0 197.41 0 1.53 0 0.02 0 1.48 0 0.01 0 143.56 0 0.35 0 50131 0 3868144 0 135116 0 6721 0 3174 0 0 0 273350 0 912 0 0 0 8269 0 1161613 0 8432 0 1179226 0 89.18 0 3449783 0 44101 1183611 26.838643114669 3868144.0 3584899.0 50131.0 135116.0 6721.0 3174.0 0.0 273350.0 3449783.0 92.7 1.3 3.5 0.2 0.1 0.0 7.1 89.2 100 100 100.00 38 386814400 27.6 21.9 21.7 28.8 0.0 35.9 19.0 smartseq 124591 SRR2049357 SRP059035 SRS951806 SRX1047437 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702605: H358_SC42; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702605 GSM1702605 H358_SC42 1213680600 6068403 2015-06-05 16:18:03 795101481 1213680600 6068403 2 6068403 index:0,count:6068403,average:100,stdev:0|index:1,count:6068403,average:100,stdev:0 GSM1702605_r1 3.51 3.35 0.13 932332945 1173648300 898440822 1136158767 125.88 126.46 5359922 4773107 258.878 1395.779 175 21146 61.32 63.81 5749505 3286553 5749505 3286553 60.37 60.52 5749505 3235740 5749505 3117098 184670453 19.81 1.75 0 3.45 0 0.15 0 0.07 0 0.00 0 11.46 0 5359922 0 200 0 196.28 0 1.52 0 0.02 0 1.47 0 0.01 0 128.51 0 0.40 0 106293 0 6068403 0 209244 0 8889 0 4306 0 0 0 695286 0 922 0 0 0 10994 0 1447504 0 13204 0 1472624 0 84.88 0 5150678 0 58302 1417257 24.308891633220 6068403.0 5359922.0 106293.0 209244.0 8889.0 4306.0 0.0 695286.0 5150678.0 88.3 1.8 3.4 0.1 0.1 0.0 11.5 84.9 100 100 100.00 38 606840300 27.5 22.2 21.5 28.8 0.0 35.8 18.7 smartseq 124593 SRR2049358 SRP059035 SRS951805 SRX1047438 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702606: H358_SC44; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702606 GSM1702606 H358_SC44 986833600 4934168 2015-06-05 16:18:03 648440119 986833600 4934168 2 4934168 index:0,count:4934168,average:100,stdev:0|index:1,count:4934168,average:100,stdev:0 GSM1702606_r1 2.66 4.68 0.12 790128831 1007055959 759416722 972674698 127.45 128.08 4476230 3859197 267.195 1384.616 163 18017 68.24 71.16 4801958 3054658 4801958 3054658 67.24 67.44 4801958 3009795 4801958 2895028 125486500 15.88 1.42 0 3.73 0 0.18 0 0.07 0 0.00 0 9.03 0 4476230 0 200 0 196.80 0 1.55 0 0.02 0 1.49 0 0.01 0 197.37 0 0.34 0 70064 0 4934168 0 183802 0 8870 0 3676 0 0 0 445392 0 1038 0 0 0 11374 0 1554056 0 10834 0 1577302 0 86.99 0 4292428 0 49494 1528255 30.877581120944 4934168.0 4476230.0 70064.0 183802.0 8870.0 3676.0 0.0 445392.0 4292428.0 90.7 1.4 3.7 0.2 0.1 0.0 9.0 87.0 100 100 100.00 38 493416800 27.4 22.1 21.7 28.7 0.0 35.9 18.9 smartseq 124595 SRR2049359 SRP059035 SRS951804 SRX1047439 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702607: H358_SC52; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702607 GSM1702607 H358_SC52 863948600 4319743 2015-06-05 16:18:03 568421129 863948600 4319743 2 4319743 index:0,count:4319743,average:100,stdev:0|index:1,count:4319743,average:100,stdev:0 GSM1702607_r1 3.23 3.34 0.09 692896944 881219486 667929410 853406903 127.18 127.77 3916569 3422112 268.411 1344.002 174 15740 68.06 70.77 4175187 2665737 4175187 2665737 66.81 67.02 4175187 2616497 4175187 2524745 109169232 15.76 1.48 0 3.46 0 0.16 0 0.06 0 0.00 0 9.12 0 3916569 0 200 0 196.85 0 1.53 0 0.02 0 1.46 0 0.01 0 132.92 0 0.34 0 63955 0 4319743 0 149672 0 6763 0 2429 0 0 0 393982 0 860 0 0 0 8359 0 1216126 0 8354 0 1233699 0 87.20 0 3766897 0 46481 1196981 25.752049224414 4319743.0 3916569.0 63955.0 149672.0 6763.0 2429.0 0.0 393982.0 3766897.0 90.7 1.5 3.5 0.2 0.1 0.0 9.1 87.2 100 100 100.00 38 431974300 27.8 21.6 21.3 29.3 0.0 35.8 18.6 smartseq 124609 SRR2049360 SRP059035 SRS951803 SRX1047440 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702608: H358_SC53; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702608 GSM1702608 H358_SC53 1194825400 5974127 2015-06-05 16:18:03 778384673 1194825400 5974127 2 5974127 index:0,count:5974127,average:100,stdev:0|index:1,count:5974127,average:100,stdev:0 GSM1702608_r1 3.14 3.61 0.12 810653935 1025633788 776836037 987125221 126.52 127.07 4827923 4256929 254.148 1314.976 133 18351 64.77 67.81 5207312 3126901 5207312 3126901 63.98 64.0 5207312 3088875 5207312 2951064 137434681 16.95 1.41 0 3.63 0 0.23 0 0.11 0 0.00 0 18.85 0 4827923 0 200 0 195.13 0 1.52 0 0.02 0 1.47 0 0.01 0 131.14 0 0.34 0 84087 0 5974127 0 216752 0 13876 0 6373 0 0 0 1125955 0 844 0 0 0 10854 0 1464116 0 9735 0 1485549 0 77.19 0 4611171 0 38232 1401868 36.667399037456 5974127.0 4827923.0 84087.0 216752.0 13876.0 6373.0 0.0 1125955.0 4611171.0 80.8 1.4 3.6 0.2 0.1 0.0 18.8 77.2 100 100 100.00 38 597412700 27.4 22.4 21.2 29.0 0.0 35.6 17.9 smartseq 124611 SRR2049361 SRP059035 SRS951802 SRX1047441 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702609: H358_SC55; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702609 GSM1702609 H358_SC55 1099181000 5495905 2015-06-05 16:18:03 717548986 1099181000 5495905 2 5495905 index:0,count:5495905,average:100,stdev:0|index:1,count:5495905,average:100,stdev:0 GSM1702609_r1 3.36 2.76 0.14 752757551 948372511 725351427 918073128 125.99 126.57 4465621 3954425 255.885 1229.549 120 16925 64.19 66.82 4782890 2866479 4782890 2866479 63.09 63.29 4782890 2817457 4782890 2715111 135475455 18.00 1.35 0 3.20 0 0.13 0 0.05 0 0.00 0 18.57 0 4465621 0 200 0 195.35 0 1.52 0 0.02 0 1.50 0 0.01 0 197.85 0 0.33 0 74407 0 5495905 0 175707 0 6910 0 2757 0 0 0 1020617 0 871 0 0 0 9552 0 1242108 0 10087 0 1262618 0 78.06 0 4289914 0 38232 1186663 31.038475622515 5495905.0 4465621.0 74407.0 175707.0 6910.0 2757.0 0.0 1020617.0 4289914.0 81.3 1.4 3.2 0.1 0.1 0.0 18.6 78.1 100 100 100.00 38 549590500 27.7 22.2 21.0 29.0 0.0 35.7 18.2 smartseq 124613 SRR2049362 SRP059035 SRS951801 SRX1047442 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702610: H358_SC57; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702610 GSM1702610 H358_SC57 865754400 4328772 2015-06-05 16:18:03 572660769 865754400 4328772 2 4328772 index:0,count:4328772,average:100,stdev:0|index:1,count:4328772,average:100,stdev:0 GSM1702610_r1 3.21 3.19 0.09 740730491 928424565 712340257 896492822 125.34 125.85 3994751 3412899 308.963 1457.382 184 12611 63.32 65.94 4257902 2529494 4257902 2529494 62.71 62.64 4257902 2504968 4257902 2402715 143135864 19.32 1.29 0 3.67 0 0.12 0 0.06 0 0.00 0 7.53 0 3994751 0 200 0 197.39 0 1.54 0 0.02 0 1.52 0 0.01 0 167.57 0 0.37 0 55898 0 4328772 0 158760 0 5389 0 2793 0 0 0 325839 0 831 0 0 0 8068 0 1203763 0 8846 0 1221508 0 88.62 0 3835991 0 38965 1230414 31.577415629411 4328772.0 3994751.0 55898.0 158760.0 5389.0 2793.0 0.0 325839.0 3835991.0 92.3 1.3 3.7 0.1 0.1 0.0 7.5 88.6 100 100 100.00 38 432877200 27.8 21.7 21.5 29.0 0.0 35.9 18.9 smartseq 124617 SRR2049364 SRP059035 SRS952007 SRX1047444 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702612: H358_SC62; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702612 GSM1702612 H358_SC62 799565800 3997829 2015-06-05 16:18:03 521031597 799565800 3997829 2 3997829 index:0,count:3997829,average:100,stdev:0|index:1,count:3997829,average:100,stdev:0 GSM1702612_r1 2.7 3.08 0.09 624327593 780438764 601504544 755129183 125.0 125.54 3558792 3147267 266.574 1417.159 164 14406 61.96 64.51 3800104 2204932 3800104 2204932 61.04 61.19 3800104 2172165 3800104 2091689 131096123 21.00 1.89 0 3.52 0 0.18 0 0.05 0 0.00 0 10.75 0 3558792 0 200 0 196.48 0 1.54 0 0.02 0 1.50 0 0.01 0 128.50 0 0.39 0 75512 0 3997829 0 140691 0 7189 0 1929 0 0 0 429919 0 527 0 0 0 7967 0 999773 0 8901 0 1017168 0 85.50 0 3418101 0 48676 983245 20.199790451146 3997829.0 3558792.0 75512.0 140691.0 7189.0 1929.0 0.0 429919.0 3418101.0 89.0 1.9 3.5 0.2 0.0 0.0 10.8 85.5 100 100 100.00 38 399782900 27.7 21.6 21.1 29.5 0.0 35.8 18.3 smartseq 124619 SRR2049365 SRP059035 SRS952006 SRX1047445 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702613: H358_SC63; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702613 GSM1702613 H358_SC63 1207332400 6036662 2015-06-05 16:18:03 788370149 1207332400 6036662 2 6036662 index:0,count:6036662,average:100,stdev:0|index:1,count:6036662,average:100,stdev:0 GSM1702613_r1 3.26 3.47 0.11 937129235 1178347639 903850451 1142488705 125.74 126.4 5349614 4745042 262.539 1470.421 175 21169 62.98 65.5 5711439 3369411 5711439 3369411 61.81 62.1 5711439 3306823 5711439 3194607 182132165 19.44 1.80 0 3.40 0 0.20 0 0.09 0 0.00 0 11.09 0 5349614 0 200 0 196.38 0 1.51 0 0.02 0 1.47 0 0.01 0 134.15 0 0.39 0 108824 0 6036662 0 205274 0 11914 0 5402 0 0 0 669732 0 863 0 0 0 12419 0 1479614 0 13001 0 1505897 0 85.22 0 5144340 0 52195 1451010 27.799789251844 6036662.0 5349614.0 108824.0 205274.0 11914.0 5402.0 0.0 669732.0 5144340.0 88.6 1.8 3.4 0.2 0.1 0.0 11.1 85.2 100 100 100.00 38 603666200 27.7 21.8 21.3 29.2 0.0 35.8 18.4 smartseq 124621 SRR2049366 SRP059035 SRS952004 SRX1047446 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702614: H358_SC65; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702614 GSM1702614 H358_SC65 1543875200 7719376 2015-06-05 16:18:03 1027537989 1543875200 7719376 2 7719376 index:0,count:7719376,average:100,stdev:0|index:1,count:7719376,average:100,stdev:0 GSM1702614_r1 4.01 3.25 0.1 1224916967 1570631117 1179228682 1518656801 128.22 128.78 6962944 6054862 260.376 1358.298 175 27730 69.4 72.25 7438351 4832621 7438351 4832621 68.3 68.48 7438351 4755682 7438351 4580029 182205251 14.87 1.41 0 3.56 0 0.15 0 0.08 0 0.00 0 9.58 0 6962944 0 200 0 196.58 0 1.51 0 0.02 0 1.47 0 0.01 0 125.75 0 0.38 0 108975 0 7719376 0 274431 0 11201 0 5838 0 0 0 739393 0 1209 0 0 0 18548 0 2332841 0 16987 0 2369585 0 86.65 0 6688513 0 52778 2304443 43.662946682330 7719376.0 6962944.0 108975.0 274431.0 11201.0 5838.0 0.0 739393.0 6688513.0 90.2 1.4 3.6 0.1 0.1 0.0 9.6 86.6 100 100 100.00 38 771937600 27.6 22.0 21.6 28.7 0.0 35.9 19.0 smartseq 124623 SRR2049367 SRP059035 SRS952005 SRX1047447 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702615: H358_SC67; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702615 GSM1702615 H358_SC67 1173339400 5866697 2015-06-05 16:18:03 767231269 1173339400 5866697 2 5866697 index:0,count:5866697,average:100,stdev:0|index:1,count:5866697,average:100,stdev:0 GSM1702615_r1 3.45 3.89 0.13 811350401 999123069 782483040 968545669 123.14 123.78 4807126 4291357 258.711 1187.580 111 18017 58.61 60.96 5139729 2817528 5139729 2817528 57.56 57.68 5139729 2766862 5139729 2666139 170380646 21.00 1.45 0 3.15 0 0.14 0 0.08 0 0.00 0 17.85 0 4807126 0 200 0 195.38 0 1.53 0 0.02 0 1.49 0 0.01 0 126.47 0 0.34 0 85107 0 5866697 0 184882 0 8001 0 4482 0 0 0 1047088 0 884 0 0 0 9663 0 1265618 0 10925 0 1287090 0 78.79 0 4622244 0 39136 1211571 30.957967089125 5866697.0 4807126.0 85107.0 184882.0 8001.0 4482.0 0.0 1047088.0 4622244.0 81.9 1.5 3.2 0.1 0.1 0.0 17.8 78.8 100 100 100.00 38 586669700 27.9 22.0 20.9 29.2 0.0 35.7 18.3 smartseq 124649 SRR2049374 SRP059035 SRS951997 SRX1047454 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702622: H358_SC75; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702622 GSM1702622 H358_SC75 732517400 3662587 2015-06-05 16:18:03 483246295 732517400 3662587 2 3662587 index:0,count:3662587,average:100,stdev:0|index:1,count:3662587,average:100,stdev:0 GSM1702622_r1 2.77 3.37 0.17 333248336 412797604 316859057 394960388 123.87 124.65 2388864 2247553 165.257 973.632 100 19796 56.9 60.03 2632531 1359336 2632531 1359336 56.24 56.21 2632531 1343465 2632531 1272780 71859919 21.56 1.43 0 3.40 0 0.19 0 0.12 0 0.00 0 34.46 0 2388864 0 200 0 191.15 0 1.54 0 0.01 0 1.52 0 0.01 0 106.33 0 0.31 0 52196 0 3662587 0 124541 0 7104 0 4569 0 0 0 1262050 0 473 0 0 0 4928 0 624974 0 5499 0 635874 0 61.82 0 2264323 0 32544 536134 16.474127335300 3662587.0 2388864.0 52196.0 124541.0 7104.0 4569.0 0.0 1262050.0 2264323.0 65.2 1.4 3.4 0.2 0.1 0.0 34.5 61.8 100 100 100.00 38 366258700 27.0 24.0 21.2 27.9 0.0 35.8 19.6 smartseq 124651 SRR2049375 SRP059035 SRS951996 SRX1047455 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702623: H358_SC76; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702623 GSM1702623 H358_SC76 857280200 4286401 2015-06-05 16:18:03 564449441 857280200 4286401 2 4286401 index:0,count:4286401,average:100,stdev:0|index:1,count:4286401,average:100,stdev:0 GSM1702623_r1 4.33 3.06 0.18 739476711 940148831 715400496 912861053 127.14 127.6 3980360 3315318 314.386 1686.554 186 12168 68.07 70.46 4213623 2709474 4213623 2709474 66.79 66.91 4213623 2658454 4213623 2572871 120580229 16.31 1.33 0 3.15 0 0.13 0 0.08 0 0.00 0 6.93 0 3980360 0 200 0 197.37 0 1.53 0 0.02 0 1.46 0 0.01 0 173.38 0 0.35 0 56824 0 4286401 0 135210 0 5454 0 3609 0 0 0 296978 0 1032 0 0 0 11133 0 1318425 0 9625 0 1340215 0 89.71 0 3845150 0 46553 1337944 28.740231564024 4286401.0 3980360.0 56824.0 135210.0 5454.0 3609.0 0.0 296978.0 3845150.0 92.9 1.3 3.2 0.1 0.1 0.0 6.9 89.7 100 100 100.00 38 428640100 27.9 21.7 21.5 29.0 0.0 35.9 19.1 smartseq 124653 SRR2049376 SRP059035 SRS951994 SRX1047456 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702624: H358_SC79; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702624 GSM1702624 H358_SC79 889739200 4448696 2015-06-05 16:18:03 593442686 889739200 4448696 2 4448696 index:0,count:4448696,average:100,stdev:0|index:1,count:4448696,average:100,stdev:0 GSM1702624_r1 3.93 3.39 0.16 758083310 961188141 731614315 930826146 126.79 127.23 4110492 3477769 300.011 1519.581 180 13397 66.44 68.95 4368581 2730966 4368581 2730966 65.72 65.69 4368581 2701282 4368581 2601937 135279808 17.84 1.34 0 3.36 0 0.11 0 0.07 0 0.00 0 7.42 0 4110492 0 200 0 197.23 0 1.53 0 0.02 0 1.49 0 0.01 0 177.95 0 0.38 0 59461 0 4448696 0 149649 0 5103 0 3017 0 0 0 330084 0 967 0 0 0 9942 0 1328508 0 10157 0 1349574 0 89.03 0 3960843 0 43933 1348671 30.698358864635 4448696.0 4110492.0 59461.0 149649.0 5103.0 3017.0 0.0 330084.0 3960843.0 92.4 1.3 3.4 0.1 0.1 0.0 7.4 89.0 100 100 100.00 38 444869600 27.8 21.8 21.6 28.8 0.0 36.0 19.3 smartseq 124655 SRR2049377 SRP059035 SRS951995 SRX1047457 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702625: H358_SC80; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702625 GSM1702625 H358_SC80 1193003000 5965015 2015-06-05 16:18:03 777837643 1193003000 5965015 2 5965015 index:0,count:5965015,average:100,stdev:0|index:1,count:5965015,average:100,stdev:0 GSM1702625_r1 4.21 3.42 0.07 959399220 1213605931 923474346 1172282564 126.5 126.94 5435670 4713097 267.284 1406.595 175 21539 66.44 69.17 5803592 3611320 5803592 3611320 65.46 65.54 5803592 3558085 5803592 3421765 158652672 16.54 1.41 0 3.59 0 0.14 0 0.08 0 0.00 0 8.65 0 5435670 0 200 0 196.86 0 1.54 0 0.02 0 1.50 0 0.01 0 161.46 0 0.33 0 84092 0 5965015 0 214413 0 8161 0 4948 0 0 0 516236 0 1330 0 0 0 14382 0 1788678 0 12794 0 1817184 0 87.53 0 5221257 0 49175 1762895 35.849415353330 5965015.0 5435670.0 84092.0 214413.0 8161.0 4948.0 0.0 516236.0 5221257.0 91.1 1.4 3.6 0.1 0.1 0.0 8.7 87.5 100 100 100.00 38 596501500 27.7 22.0 21.5 28.8 0.0 36.0 19.2 smartseq 124657 SRR2049378 SRP059035 SRS951993 SRX1047458 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702626: H358_SC83; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702626 GSM1702626 H358_SC83 1145197400 5725987 2015-06-05 16:18:03 745114101 1145197400 5725987 2 5725987 index:0,count:5725987,average:100,stdev:0|index:1,count:5725987,average:100,stdev:0 GSM1702626_r1 4.09 3.82 0.12 921074274 1166522298 887384504 1128224292 126.65 127.14 5219971 4541953 266.091 1376.238 163 21111 66.26 68.91 5581741 3458575 5581741 3458575 65.29 65.45 5581741 3408376 5581741 3284819 159441093 17.31 1.37 0 3.51 0 0.14 0 0.16 0 0.00 0 8.54 0 5219971 0 200 0 196.87 0 1.54 0 0.02 0 1.49 0 0.01 0 150.46 0 0.32 0 78383 0 5725987 0 200879 0 7937 0 9053 0 0 0 489026 0 925 0 0 0 13873 0 1708652 0 12623 0 1736073 0 87.65 0 5019092 0 49597 1680684 33.886807669819 5725987.0 5219971.0 78383.0 200879.0 7937.0 9053.0 0.0 489026.0 5019092.0 91.2 1.4 3.5 0.1 0.2 0.0 8.5 87.7 100 100 100.00 38 572598700 27.6 22.1 21.7 28.7 0.0 36.0 19.3 smartseq 249123 SRR2049348 SRP059035 SRS951815 SRX1047428 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702596: H358_SC18; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702596 GSM1702596 H358_SC18 919297600 4596488 2015-06-05 16:18:03 602079235 919297600 4596488 2 4596488 index:0,count:4596488,average:100,stdev:0|index:1,count:4596488,average:100,stdev:0 GSM1702596_r1 3.4 3.19 0.11 792114244 1006564605 765042210 975502264 127.07 127.51 4270030 3523656 311.646 1685.083 175 13015 68.7 71.24 4534898 2933463 4534898 2933463 67.61 67.69 4534898 2886795 4534898 2787162 125519932 15.85 1.33 0 3.32 0 0.13 0 0.07 0 0.00 0 6.89 0 4270030 0 200 0 197.33 0 1.53 0 0.02 0 1.52 0 0.01 0 146.44 0 0.34 0 60982 0 4596488 0 152432 0 6199 0 3339 0 0 0 316920 0 925 0 0 0 12076 0 1510148 0 10435 0 1533584 0 89.58 0 4117598 0 47105 1527967 32.437469483070 4596488.0 4270030.0 60982.0 152432.0 6199.0 3339.0 0.0 316920.0 4117598.0 92.9 1.3 3.3 0.1 0.1 0.0 6.9 89.6 100 100 100.00 38 459648800 27.6 21.9 21.7 28.7 0.0 36.0 19.2 smartseq 249155 SRR2049350 SRP059035 SRS951813 SRX1047430 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702598: H358_SC24; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702598 GSM1702598 H358_SC24 1220656200 6103281 2015-06-05 16:18:03 801453493 1220656200 6103281 2 6103281 index:0,count:6103281,average:100,stdev:0|index:1,count:6103281,average:100,stdev:0 GSM1702598_r1 2.54 3.55 0.17 847012496 1025421161 817847124 997170879 121.06 121.93 5001410 4485897 259.413 1534.911 111 18682 57.55 59.76 5345224 2878250 5345224 2878250 56.53 56.74 5345224 2827459 5345224 2732665 189244183 22.34 1.59 0 3.03 0 0.22 0 0.16 0 0.00 0 17.68 0 5001410 0 200 0 195.40 0 1.53 0 0.02 0 1.50 0 0.01 0 127.00 0 0.35 0 97059 0 6103281 0 184880 0 13360 0 9654 0 0 0 1078857 0 1328 0 0 0 9247 0 1188133 0 11178 0 1209886 0 78.92 0 4816530 0 38443 1141894 29.703561116458 6103281.0 5001410.0 97059.0 184880.0 13360.0 9654.0 0.0 1078857.0 4816530.0 81.9 1.6 3.0 0.2 0.2 0.0 17.7 78.9 100 100 100.00 38 610328100 28.2 21.7 20.6 29.5 0.0 35.7 18.2 smartseq 249159 SRR2049351 SRP059035 SRS951812 SRX1047431 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702599: H358_SC25; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702599 GSM1702599 H358_SC25 1038895600 5194478 2015-06-05 16:18:03 686035594 1038895600 5194478 2 5194478 index:0,count:5194478,average:100,stdev:0|index:1,count:5194478,average:100,stdev:0 GSM1702599_r1 3.41 3.15 0.13 891245204 1131182773 857702134 1093566526 126.92 127.5 4806347 3988573 309.128 1647.525 187 14945 68.86 71.66 5124673 3309691 5124673 3309691 67.89 68.05 5124673 3263023 5124673 3143056 139116873 15.61 1.34 0 3.61 0 0.16 0 0.11 0 0.00 0 7.20 0 4806347 0 200 0 197.31 0 1.52 0 0.01 0 1.49 0 0.01 0 165.49 0 0.35 0 69822 0 5194478 0 187618 0 8369 0 5630 0 0 0 374132 0 1209 0 0 0 12480 0 1675652 0 11183 0 1700524 0 88.92 0 4618729 0 47933 1700305 35.472534579517 5194478.0 4806347.0 69822.0 187618.0 8369.0 5630.0 0.0 374132.0 4618729.0 92.5 1.3 3.6 0.2 0.1 0.0 7.2 88.9 100 100 100.00 38 519447800 27.8 21.7 21.6 28.9 0.0 35.9 19.1 smartseq 249162 SRR2049352 SRP059035 SRS951811 SRX1047432 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702600: H358_SC29; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702600 GSM1702600 H358_SC29 1165895800 5829479 2015-06-05 16:18:03 764783778 1165895800 5829479 2 5829479 index:0,count:5829479,average:100,stdev:0|index:1,count:5829479,average:100,stdev:0 GSM1702600_r1 4.34 3.4 0.15 803092854 1062729280 767667771 1021380807 132.33 133.05 4771158 4053042 257.194 1454.281 111 17331 75.95 79.75 5174928 3623664 5174928 3623664 74.88 75.29 5174928 3572832 5174928 3420818 87317306 10.87 1.37 0 3.90 0 0.19 0 0.05 0 0.00 0 17.92 0 4771158 0 200 0 195.09 0 1.51 0 0.01 0 1.43 0 0.01 0 131.16 0 0.34 0 79942 0 5829479 0 227339 0 10934 0 2949 0 0 0 1044438 0 1239 0 0 0 12059 0 1766091 0 12407 0 1791796 0 77.95 0 4543819 0 45217 1685237 37.269986951810 5829479.0 4771158.0 79942.0 227339.0 10934.0 2949.0 0.0 1044438.0 4543819.0 81.8 1.4 3.9 0.2 0.1 0.0 17.9 77.9 100 100 100.00 38 582947900 27.2 22.7 21.6 28.4 0.0 35.7 18.5 smartseq 249166 SRR2049353 SRP059035 SRS951810 SRX1047433 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702601: H358_SC32; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702601 GSM1702601 H358_SC32 849455400 4247277 2015-06-05 16:18:03 566534098 849455400 4247277 2 4247277 index:0,count:4247277,average:100,stdev:0|index:1,count:4247277,average:100,stdev:0 GSM1702601_r1 4.07 3.13 0.1 719473068 912119074 691696258 880652779 126.78 127.32 3903996 3315522 298.204 1524.717 183 12767 67.28 70.09 4172064 2626573 4172064 2626573 66.6 66.64 4172064 2599999 4172064 2497269 116116669 16.14 1.31 0 3.69 0 0.16 0 0.13 0 0.00 0 7.80 0 3903996 0 200 0 197.15 0 1.54 0 0.02 0 1.45 0 0.01 0 149.90 0 0.39 0 55814 0 4247277 0 156545 0 6610 0 5519 0 0 0 331152 0 750 0 0 0 9611 0 1254567 0 8853 0 1273781 0 88.23 0 3747451 0 41714 1280163 30.689049240063 4247277.0 3903996.0 55814.0 156545.0 6610.0 5519.0 0.0 331152.0 3747451.0 91.9 1.3 3.7 0.2 0.1 0.0 7.8 88.2 100 100 100.00 38 424727700 27.9 21.6 21.5 28.9 0.0 35.9 19.1 smartseq 249170 SRR2049354 SRP059035 SRS951809 SRX1047434 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702602: H358_SC34; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702602 GSM1702602 H358_SC34 1272097600 6360488 2015-06-05 16:18:03 829974157 1272097600 6360488 2 6360488 index:0,count:6360488,average:100,stdev:0|index:1,count:6360488,average:100,stdev:0 GSM1702602_r1 4.3 3.31 0.08 838504845 1074969800 808773245 1041664596 128.2 128.8 5059567 4395904 242.977 1354.576 101 20619 68.25 70.97 5426216 3453118 5426216 3453118 66.96 67.25 5426216 3387890 5426216 3271861 137415717 16.39 1.43 0 3.05 0 0.14 0 0.08 0 0.00 0 20.23 0 5059567 0 200 0 194.76 0 1.52 0 0.01 0 1.45 0 0.01 0 136.30 0 0.33 0 90848 0 6360488 0 194261 0 9101 0 4997 0 0 0 1286823 0 1193 0 0 0 12160 0 1674379 0 13244 0 1700976 0 76.49 0 4865306 0 42089 1577122 37.471120720378 6360488.0 5059567.0 90848.0 194261.0 9101.0 4997.0 0.0 1286823.0 4865306.0 79.5 1.4 3.1 0.1 0.1 0.0 20.2 76.5 100 100 100.00 38 636048800 27.3 22.8 21.3 28.6 0.0 35.7 18.4 smartseq 249230 SRR2049363 SRP059035 SRS951800 SRX1047443 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702611: H358_SC59; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702611 GSM1702611 H358_SC59 1103032400 5515162 2015-06-05 16:18:03 731180631 1103032400 5515162 2 5515162 index:0,count:5515162,average:100,stdev:0|index:1,count:5515162,average:100,stdev:0 GSM1702611_r1 4.13 3.52 0.1 880097644 1124278510 851251129 1091239996 127.74 128.19 5007295 4386773 258.886 1280.107 176 20903 67.41 69.83 5309087 3375610 5309087 3375610 65.86 66.01 5309087 3297833 5309087 3191144 141196347 16.04 1.33 0 3.14 0 0.11 0 0.12 0 0.00 0 8.98 0 5007295 0 200 0 196.81 0 1.53 0 0.02 0 1.49 0 0.01 0 180.50 0 0.36 0 73497 0 5515162 0 173238 0 6001 0 6867 0 0 0 494999 0 1024 0 0 0 11634 0 1607065 0 11752 0 1631475 0 87.65 0 4834057 0 47454 1568153 33.045749568003 5515162.0 5007295.0 73497.0 173238.0 6001.0 6867.0 0.0 494999.0 4834057.0 90.8 1.3 3.1 0.1 0.1 0.0 9.0 87.7 100 100 100.00 38 551516200 27.6 22.1 21.7 28.7 0.0 35.9 19.0 smartseq 249250 SRR2049368 SRP059035 SRS952003 SRX1047448 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702616: H358_SC68; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702616 GSM1702616 H358_SC68 1297477800 6487389 2015-06-05 16:18:03 863868695 1297477800 6487389 2 6487389 index:0,count:6487389,average:100,stdev:0|index:1,count:6487389,average:100,stdev:0 GSM1702616_r1 4.33 3.14 0.17 862894340 1079776971 832405483 1044787824 125.13 125.51 5199976 4617599 243.130 1243.999 100 21317 61.93 64.38 5549506 3220238 5549506 3220238 61.07 61.0 5549506 3175376 5549506 3051204 174348855 20.21 1.32 0 3.06 0 0.12 0 0.06 0 0.00 0 19.66 0 5199976 0 200 0 194.98 0 1.55 0 0.02 0 1.47 0 0.01 0 185.35 0 0.37 0 85740 0 6487389 0 198391 0 7889 0 3955 0 0 0 1275569 0 1155 0 0 0 11758 0 1488871 0 11040 0 1512824 0 77.10 0 5001585 0 39690 1409697 35.517687074830 6487389.0 5199976.0 85740.0 198391.0 7889.0 3955.0 0.0 1275569.0 5001585.0 80.2 1.3 3.1 0.1 0.1 0.0 19.7 77.1 100 100 100.00 38 648738900 27.6 22.6 21.1 28.8 0.0 35.8 18.7 smartseq 249254 SRR2049369 SRP059035 SRS952002 SRX1047449 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702617: H358_SC69; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702617 GSM1702617 H358_SC69 1051158800 5255794 2015-06-05 16:18:03 685611753 1051158800 5255794 2 5255794 index:0,count:5255794,average:100,stdev:0|index:1,count:5255794,average:100,stdev:0 GSM1702617_r1 3.06 3.57 0.18 721570904 895854158 695495746 866666480 124.15 124.61 4289858 3816865 251.600 1272.606 110 16363 60.38 62.81 4585856 2590140 4585856 2590140 59.54 59.48 4585856 2554317 4585856 2452870 147798514 20.48 1.42 0 3.16 0 0.15 0 0.10 0 0.00 0 18.13 0 4289858 0 200 0 195.30 0 1.53 0 0.02 0 1.51 0 0.01 0 159.00 0 0.33 0 74592 0 5255794 0 166169 0 7640 0 5318 0 0 0 952978 0 533 0 0 0 8010 0 1166525 0 9628 0 1184696 0 78.46 0 4123689 0 36220 1122119 30.980646051905 5255794.0 4289858.0 74592.0 166169.0 7640.0 5318.0 0.0 952978.0 4123689.0 81.6 1.4 3.2 0.1 0.1 0.0 18.1 78.5 100 100 100.00 38 525579400 27.7 22.3 21.0 29.0 0.0 35.8 18.3 smartseq 249282 SRR2049370 SRP059035 SRS952001 SRX1047450 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702618: H358_SC70; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702618 GSM1702618 H358_SC70 938999000 4694995 2015-06-05 16:18:03 624324868 938999000 4694995 2 4694995 index:0,count:4694995,average:100,stdev:0|index:1,count:4694995,average:100,stdev:0 GSM1702618_r1 4.61 3.27 0.08 748291473 1009087176 715432005 969886255 134.85 135.57 4265173 3525067 264.011 1503.021 174 17283 82.99 87.04 4604840 3539823 4604840 3539823 81.66 82.14 4604840 3483052 4604840 3340748 46615956 6.23 1.35 0 4.22 0 0.16 0 0.09 0 0.00 0 8.90 0 4265173 0 200 0 196.46 0 1.48 0 0.01 0 1.50 0 0.01 0 138.54 0 0.35 0 63168 0 4694995 0 198092 0 7495 0 4256 0 0 0 418071 0 1082 0 0 0 13215 0 1919231 0 10372 0 1943900 0 86.63 0 4067081 0 49573 1874673 37.816412159845 4694995.0 4265173.0 63168.0 198092.0 7495.0 4256.0 0.0 418071.0 4067081.0 90.8 1.3 4.2 0.2 0.1 0.0 8.9 86.6 100 100 100.00 38 469499500 26.8 22.8 22.4 28.0 0.0 35.8 19.2 smartseq 249286 SRR2049371 SRP059035 SRS952000 SRX1047451 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702619: H358_SC71; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702619 GSM1702619 H358_SC71 1621209800 8106049 2015-06-05 16:18:03 1060082037 1621209800 8106049 2 8106049 index:0,count:8106049,average:100,stdev:0|index:1,count:8106049,average:100,stdev:0 GSM1702619_r1 3.28 3.28 0.1 1295612161 1632714750 1245973525 1576660843 126.02 126.54 7340625 6400989 264.513 1326.805 175 28911 64.5 67.21 7826387 4734629 7826387 4734629 63.63 63.58 7826387 4671149 7826387 4478851 240865473 18.59 1.44 0 3.65 0 0.15 0 0.07 0 0.00 0 9.23 0 7340625 0 200 0 196.69 0 1.53 0 0.01 0 1.50 0 0.01 0 173.70 0 0.34 0 116384 0 8106049 0 296251 0 11986 0 5507 0 0 0 747931 0 1571 0 0 0 16820 0 2387576 0 16659 0 2422626 0 86.90 0 7044374 0 50759 2352544 46.347327567525 8106049.0 7340625.0 116384.0 296251.0 11986.0 5507.0 0.0 747931.0 7044374.0 90.6 1.4 3.7 0.1 0.1 0.0 9.2 86.9 100 100 100.00 38 810604900 27.7 21.9 21.6 28.8 0.0 36.0 19.2 smartseq 249291 SRR2049372 SRP059035 SRS951999 SRX1047452 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702620: H358_SC73; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702620 GSM1702620 H358_SC73 962851200 4814256 2015-06-05 16:18:03 634286486 962851200 4814256 2 4814256 index:0,count:4814256,average:100,stdev:0|index:1,count:4814256,average:100,stdev:0 GSM1702620_r1 3.94 3.51 0.12 666140701 841352789 640880456 813099855 126.3 126.87 3955606 3456096 253.578 1316.308 133 14754 65.76 68.57 4231143 2601315 4231143 2601315 64.84 64.96 4231143 2564690 4231143 2464248 114582103 17.20 1.41 0 3.36 0 0.15 0 0.08 0 0.00 0 17.61 0 3955606 0 200 0 195.24 0 1.53 0 0.02 0 1.50 0 0.01 0 165.06 0 0.34 0 67705 0 4814256 0 161831 0 7166 0 3890 0 0 0 847594 0 737 0 0 0 8917 0 1238273 0 9410 0 1257337 0 78.80 0 3793775 0 37417 1180597 31.552422695566 4814256.0 3955606.0 67705.0 161831.0 7166.0 3890.0 0.0 847594.0 3793775.0 82.2 1.4 3.4 0.1 0.1 0.0 17.6 78.8 100 100 100.00 38 481425600 27.6 22.4 21.1 28.9 0.0 35.7 18.4 smartseq 249295 SRR2049373 SRP059035 SRS951998 SRX1047453 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702621: H358_SC74; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702621 GSM1702621 H358_SC74 986609200 4933046 2015-06-05 16:18:03 648330556 986609200 4933046 2 4933046 index:0,count:4933046,average:100,stdev:0|index:1,count:4933046,average:100,stdev:0 GSM1702621_r1 3.82 3.62 0.12 842501229 1045284697 813422953 1012987579 124.07 124.53 4549461 3934716 306.224 1433.752 195 14382 61.03 63.31 4838114 2776706 4838114 2776706 60.51 60.45 4838114 2752921 4838114 2651479 171715932 20.38 1.34 0 3.31 0 0.14 0 0.08 0 0.00 0 7.56 0 4549461 0 200 0 197.41 0 1.54 0 0.02 0 1.50 0 0.01 0 153.09 0 0.34 0 66323 0 4933046 0 163518 0 6732 0 3887 0 0 0 372966 0 842 0 0 0 9975 0 1273702 0 10064 0 1294583 0 88.91 0 4385943 0 39366 1299882 33.020423715897 4933046.0 4549461.0 66323.0 163518.0 6732.0 3887.0 0.0 372966.0 4385943.0 92.2 1.3 3.3 0.1 0.1 0.0 7.6 88.9 100 100 100.00 38 493304600 28.1 21.5 21.3 29.2 0.0 36.0 19.1 smartseq 249318 SRR2049379 SRP059035 SRS951992 SRX1047459 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702627: H358_SC84; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702627 GSM1702627 H358_SC84 946062600 4730313 2015-06-05 16:18:03 618075799 946062600 4730313 2 4730313 index:0,count:4730313,average:100,stdev:0|index:1,count:4730313,average:100,stdev:0 GSM1702627_r1 3.49 3.17 0.14 760991969 960773870 733073166 928988531 126.25 126.73 4303724 3737760 270.473 1348.754 162 17057 64.27 66.86 4600091 2765999 4600091 2765999 63.26 63.3 4600091 2722419 4600091 2618774 143325250 18.83 1.52 0 3.52 0 0.16 0 0.09 0 0.00 0 8.77 0 4303724 0 200 0 196.83 0 1.54 0 0.02 0 1.49 0 0.01 0 163.74 0 0.34 0 71689 0 4730313 0 166707 0 7477 0 4492 0 0 0 414620 0 973 0 0 0 11281 0 1407991 0 10602 0 1430847 0 87.46 0 4137017 0 47064 1389131 29.515787013429 4730313.0 4303724.0 71689.0 166707.0 7477.0 4492.0 0.0 414620.0 4137017.0 91.0 1.5 3.5 0.2 0.1 0.0 8.8 87.5 100 100 100.00 38 473031300 27.5 22.1 21.7 28.7 0.0 35.9 19.0 smartseq 249346 SRR2049380 SRP059035 SRS951991 SRX1047460 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702628: H358_SC85; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702628 GSM1702628 H358_SC85 1253770800 6268854 2015-06-05 16:18:03 818864628 1253770800 6268854 2 6268854 index:0,count:6268854,average:100,stdev:0|index:1,count:6268854,average:100,stdev:0 GSM1702628_r1 3.0 3.43 0.09 1009940440 1248364923 971780612 1205117692 123.61 124.01 5720495 4999964 265.903 1279.847 175 22913 61.6 64.15 6100370 3524100 6100370 3524100 60.93 60.74 6100370 3485229 6100370 3336923 201189666 19.92 1.39 0 3.61 0 0.11 0 0.11 0 0.00 0 8.54 0 5720495 0 200 0 196.92 0 1.56 0 0.02 0 1.51 0 0.01 0 208.96 0 0.33 0 87415 0 6268854 0 226537 0 6653 0 6655 0 0 0 535051 0 1309 0 0 0 14095 0 1818921 0 13435 0 1847760 0 87.64 0 5493958 0 45920 1797044 39.134233449477 6268854.0 5720495.0 87415.0 226537.0 6653.0 6655.0 0.0 535051.0 5493958.0 91.3 1.4 3.6 0.1 0.1 0.0 8.5 87.6 100 100 100.00 38 626885400 27.7 21.9 21.5 28.8 0.0 36.0 19.3 smartseq 249351 SRR2049381 SRP059035 SRS951990 SRX1047461 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702629: H358_SC86; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702629 GSM1702629 H358_SC86 1865471800 9327359 2015-06-05 16:18:03 1227443221 1865471800 9327359 2 9327359 index:0,count:9327359,average:100,stdev:0|index:1,count:9327359,average:100,stdev:0 GSM1702629_r1 2.74 2.9 0.08 1489275286 1891010369 1431004849 1825035677 126.98 127.54 8450831 7287959 264.739 1378.832 176 33103 68.91 71.87 9057656 5823854 9057656 5823854 67.91 68.09 9057656 5738692 9057656 5517478 223858843 15.03 1.37 0 3.72 0 0.19 0 0.13 0 0.00 0 9.08 0 8450831 0 200 0 196.66 0 1.52 0 0.01 0 1.47 0 0.01 0 154.74 0 0.34 0 127527 0 9327359 0 347425 0 17818 0 11727 0 0 0 846983 0 1744 0 0 0 22252 0 2965355 0 21727 0 3011078 0 86.88 0 8103406 0 53972 2915714 54.022715482102 9327359.0 8450831.0 127527.0 347425.0 17818.0 11727.0 0.0 846983.0 8103406.0 90.6 1.4 3.7 0.2 0.1 0.0 9.1 86.9 100 100 100.00 38 932735900 27.5 22.3 21.9 28.4 0.0 35.9 19.3 smartseq 249355 SRR2049382 SRP059035 SRS951988 SRX1047462 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702630: H358_SC87; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702630 GSM1702630 H358_SC87 947093800 4735469 2015-06-05 16:18:03 620211229 947093800 4735469 2 4735469 index:0,count:4735469,average:100,stdev:0|index:1,count:4735469,average:100,stdev:0 GSM1702630_r1 3.75 3.19 0.11 819055517 1036264070 787257305 998875631 126.52 126.88 4410483 3686004 312.963 1574.554 197 13535 65.44 68.18 4698724 2886102 4698724 2886102 64.88 64.72 4698724 2861366 4698724 2739682 146121386 17.84 1.22 0 3.75 0 0.11 0 0.07 0 0.00 0 6.68 0 4410483 0 200 0 197.43 0 1.54 0 0.01 0 1.51 0 0.01 0 193.72 0 0.33 0 57971 0 4735469 0 177416 0 5216 0 3417 0 0 0 316353 0 963 0 0 0 11309 0 1496728 0 10953 0 1519953 0 89.39 0 4233067 0 42195 1531732 36.301267922740 4735469.0 4410483.0 57971.0 177416.0 5216.0 3417.0 0.0 316353.0 4233067.0 93.1 1.2 3.7 0.1 0.1 0.0 6.7 89.4 100 100 100.00 38 473546900 27.7 21.9 21.7 28.7 0.0 36.0 19.3 smartseq 249358 SRR2049383 SRP059035 SRS951989 SRX1047463 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702631: H358_SC89; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702631 GSM1702631 H358_SC89 922843800 4614219 2015-06-05 16:18:03 605322818 922843800 4614219 2 4614219 index:0,count:4614219,average:100,stdev:0|index:1,count:4614219,average:100,stdev:0 GSM1702631_r1 2.92 3.03 0.14 794929445 984394206 769585803 957027486 123.83 124.36 4281785 3658176 311.085 1547.927 186 13185 61.8 63.93 4530668 2646179 4530668 2646179 60.87 60.9 4530668 2606491 4530668 2520809 165344436 20.80 1.28 0 3.08 0 0.13 0 0.08 0 0.00 0 7.00 0 4281785 0 200 0 197.50 0 1.54 0 0.02 0 1.48 0 0.01 0 193.15 0 0.34 0 58858 0 4614219 0 142312 0 5871 0 3502 0 0 0 323061 0 872 0 0 0 8979 0 1260501 0 10102 0 1280454 0 89.71 0 4139473 0 43169 1279543 29.640320600431 4614219.0 4281785.0 58858.0 142312.0 5871.0 3502.0 0.0 323061.0 4139473.0 92.8 1.3 3.1 0.1 0.1 0.0 7.0 89.7 100 100 100.00 38 461421900 28.0 21.6 21.3 29.1 0.0 36.0 19.2 smartseq 249362 SRR2049384 SRP059035 SRS951987 SRX1047464 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702632: H358_SC90; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702632 GSM1702632 H358_SC90 1271506400 6357532 2015-06-05 16:18:03 835499261 1271506400 6357532 2 6357532 index:0,count:6357532,average:100,stdev:0|index:1,count:6357532,average:100,stdev:0 GSM1702632_r1 3.11 3.55 0.13 840155813 1043955258 809035286 1010476675 124.26 124.9 5060184 4596674 243.988 1212.629 100 21151 57.78 60.17 5445044 2923821 5445044 2923821 57.02 57.0 5445044 2885504 5445044 2770043 184729403 21.99 1.43 0 3.16 0 0.17 0 0.13 0 0.00 0 20.11 0 5060184 0 200 0 195.00 0 1.53 0 0.02 0 1.49 0 0.01 0 137.87 0 0.34 0 90924 0 6357532 0 200628 0 10521 0 8142 0 0 0 1278685 0 683 0 0 0 8128 0 1187169 0 11199 0 1207179 0 76.44 0 4859556 0 35771 1131712 31.637695339800 6357532.0 5060184.0 90924.0 200628.0 10521.0 8142.0 0.0 1278685.0 4859556.0 79.6 1.4 3.2 0.2 0.1 0.0 20.1 76.4 100 100 100.00 38 635753200 27.6 22.4 20.9 29.1 0.0 35.7 18.2 smartseq 249366 SRR2049385 SRP059035 SRS951986 SRX1047465 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702633: H358_SC91; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702633 GSM1702633 H358_SC91 1612303400 8061517 2015-06-05 16:18:03 1055069084 1612303400 8061517 2 8061517 index:0,count:8061517,average:100,stdev:0|index:1,count:8061517,average:100,stdev:0 GSM1702633_r1 3.71 3.22 0.09 1287461268 1622010755 1242849549 1573618776 125.99 126.61 7279349 6383903 265.552 1376.346 175 28411 66.15 68.67 7750460 4815637 7750460 4815637 64.85 65.1 7750460 4720734 7750460 4565465 219666959 17.06 1.50 0 3.31 0 0.14 0 0.08 0 0.00 0 9.48 0 7279349 0 200 0 196.73 0 1.52 0 0.02 0 1.50 0 0.01 0 184.85 0 0.34 0 120906 0 8061517 0 266859 0 11575 0 6231 0 0 0 764362 0 1778 0 0 0 16479 0 2215743 0 16147 0 2250147 0 86.99 0 7012490 0 52201 2181553 41.791402463554 8061517.0 7279349.0 120906.0 266859.0 11575.0 6231.0 0.0 764362.0 7012490.0 90.3 1.5 3.3 0.1 0.1 0.0 9.5 87.0 100 100 100.00 38 806151700 27.9 21.7 21.3 29.1 0.0 35.9 18.9 smartseq 249370 SRR2049386 SRP059035 SRS951985 SRX1047466 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702634: H358_SC93; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702634 GSM1702634 H358_SC93 1377254000 6886270 2015-06-05 16:18:03 913387704 1377254000 6886270 2 6886270 index:0,count:6886270,average:100,stdev:0|index:1,count:6886270,average:100,stdev:0 GSM1702634_r1 4.04 3.22 0.14 1081636729 1358040666 1041980699 1314167487 125.55 126.12 6167552 5484358 257.977 1306.696 176 24860 63.71 66.28 6601561 3929614 6601561 3929614 62.62 62.73 6601561 3861857 6601561 3719038 196527455 18.17 1.49 0 3.47 0 0.17 0 0.12 0 0.00 0 10.15 0 6167552 0 200 0 196.58 0 1.55 0 0.02 0 1.49 0 0.01 0 193.68 0 0.38 0 102764 0 6886270 0 239168 0 11523 0 8296 0 0 0 698899 0 1088 0 0 0 13058 0 1746449 0 13639 0 1774234 0 86.09 0 5928384 0 48889 1716434 35.108797480006 6886270.0 6167552.0 102764.0 239168.0 11523.0 8296.0 0.0 698899.0 5928384.0 89.6 1.5 3.5 0.2 0.1 0.0 10.1 86.1 100 100 100.00 38 688627000 27.9 21.7 21.2 29.2 0.0 35.9 18.8 smartseq 249374 SRR2049387 SRP059035 SRS951984 SRX1047467 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702635: H358_SC94; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702635 GSM1702635 H358_SC94 1008138200 5040691 2015-06-05 16:18:03 662415901 1008138200 5040691 2 5040691 index:0,count:5040691,average:100,stdev:0|index:1,count:5040691,average:100,stdev:0 GSM1702635_r1 3.41 3.06 0.14 864867588 1070893683 837657203 1041354025 123.82 124.32 4654599 3983554 312.148 1578.419 195 14232 60.9 62.97 4919340 2834741 4919340 2834741 59.98 59.95 4919340 2792017 4919340 2698919 179643037 20.77 1.36 0 3.03 0 0.13 0 0.09 0 0.00 0 7.44 0 4654599 0 200 0 197.50 0 1.55 0 0.02 0 1.50 0 0.01 0 179.67 0 0.36 0 68447 0 5040691 0 152514 0 6374 0 4644 0 0 0 375074 0 842 0 0 0 11116 0 1334932 0 10911 0 1357801 0 89.31 0 4502085 0 43756 1363532 31.162172044977 5040691.0 4654599.0 68447.0 152514.0 6374.0 4644.0 0.0 375074.0 4502085.0 92.3 1.4 3.0 0.1 0.1 0.0 7.4 89.3 100 100 100.00 38 504069100 28.0 21.6 21.4 29.1 0.0 36.0 19.2 smartseq 249378 SRR2049388 SRP059035 SRS951980 SRX1047468 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702636: H358_SC95; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702636 GSM1702636 H358_SC95 1175208400 5876042 2015-06-05 16:18:03 763798322 1175208400 5876042 2 5876042 index:0,count:5876042,average:100,stdev:0|index:1,count:5876042,average:100,stdev:0 GSM1702636_r1 4.69 3.33 0.11 935395908 1203038071 900896665 1164087696 128.61 129.21 5320877 4648009 262.475 1351.068 163 21731 68.69 71.47 5695941 3654943 5695941 3654943 67.43 67.69 5695941 3587722 5695941 3461424 141476144 15.12 1.45 0 3.53 0 0.14 0 0.12 0 0.00 0 9.19 0 5320877 0 200 0 196.73 0 1.51 0 0.02 0 1.45 0 0.01 0 152.19 0 0.33 0 85348 0 5876042 0 207154 0 7962 0 7301 0 0 0 539902 0 864 0 0 0 13559 0 1709743 0 11932 0 1736098 0 87.03 0 5113723 0 47194 1679948 35.596643641141 5876042.0 5320877.0 85348.0 207154.0 7962.0 7301.0 0.0 539902.0 5113723.0 90.6 1.5 3.5 0.1 0.1 0.0 9.2 87.0 100 100 100.00 38 587604200 27.7 21.9 21.5 29.0 0.0 35.9 18.8 smartseq 249382 SRR2049389 SRP059035 SRS951981 SRX1047469 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702637: H358_SC96; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702637 GSM1702637 H358_SC96 1184570600 5922853 2015-06-05 16:18:03 772290521 1184570600 5922853 2 5922853 index:0,count:5922853,average:100,stdev:0|index:1,count:5922853,average:100,stdev:0 GSM1702637_r1 3.53 3.58 0.1 948499862 1191392453 915141690 1154737301 125.61 126.18 5375686 4727188 267.217 1298.503 174 21596 63.56 66.02 5729322 3416740 5729322 3416740 62.46 62.6 5729322 3357876 5729322 3239696 178738035 18.84 1.57 0 3.38 0 0.13 0 0.08 0 0.00 0 9.03 0 5375686 0 200 0 196.86 0 1.52 0 0.02 0 1.48 0 0.01 0 159.12 0 0.33 0 92822 0 5922853 0 200047 0 7886 0 4542 0 0 0 534739 0 1080 0 0 0 13375 0 1617355 0 12853 0 1644663 0 87.38 0 5175639 0 47969 1595382 33.258604515416 5922853.0 5375686.0 92822.0 200047.0 7886.0 4542.0 0.0 534739.0 5175639.0 90.8 1.6 3.4 0.1 0.1 0.0 9.0 87.4 100 100 100.00 38 592285300 27.7 21.9 21.5 28.9 0.0 35.9 19.0 smartseq 249410 SRR2049390 SRP059035 SRS951983 SRX1047470 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702638: LC-PT-45_BKM120; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702638 GSM1702638 LC-PT-45_BKM120 466963000 2334815 2015-06-05 16:18:03 268814996 466963000 2334815 2 2334815 index:0,count:2334815,average:100,stdev:0|index:1,count:2334815,average:100,stdev:0 GSM1702638_r1 1.01 2.72 0.04 434117883 591722353 414132496 569560904 136.3 137.53 2238131 1440004 396.033 3066.811 272 4214 91.0 95.46 2491050 2036707 2491050 2036707 90.11 90.92 2491050 2016717 2491050 1939876 9404379 2.17 0.98 0 4.48 0 0.07 0 0.01 0 0.00 0 4.06 0 2238131 0 200 0 197.31 0 1.55 0 0.01 0 1.53 0 0.01 0 182.72 0 0.29 0 22871 0 2334815 0 104616 0 1536 0 280 0 0 0 94868 0 535 0 0 0 9508 0 1263393 0 5339 0 1278775 0 91.38 0 2133515 0 92199 1305038 14.154578683066 2334815.0 2238131.0 22871.0 104616.0 1536.0 280.0 0.0 94868.0 2133515.0 95.9 1.0 4.5 0.1 0.0 0.0 4.1 91.4 100 100 100.00 38 233481500 25.8 23.9 23.9 26.4 0.0 37.2 21.6 smartseq 249414 SRR2049391 SRP059035 SRS951982 SRX1047471 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702639: LC-PT-45_Carboplatin; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702639 GSM1702639 LC-PT-45_Carboplatin 434663600 2173318 2015-06-05 16:18:03 250558780 434663600 2173318 2 2173318 index:0,count:2173318,average:100,stdev:0|index:1,count:2173318,average:100,stdev:0 GSM1702639_r1 0.84 2.78 0.04 402637707 547766968 385163943 527770910 136.04 137.03 2074634 1324452 399.316 3158.644 280 3823 91.73 95.98 2274379 1903089 2274379 1903089 90.8 91.5 2274379 1883729 2274379 1814200 7656130 1.90 1.22 0 4.22 0 0.08 0 0.01 0 0.00 0 4.46 0 2074634 0 200 0 197.23 0 1.55 0 0.01 0 1.52 0 0.01 0 128.26 0 0.30 0 26583 0 2173318 0 91794 0 1644 0 199 0 0 0 96841 0 540 0 0 0 8915 0 1171782 0 5367 0 1186604 0 91.24 0 1982840 0 92219 1211270 13.134711935718 2173318.0 2074634.0 26583.0 91794.0 1644.0 199.0 0.0 96841.0 1982840.0 95.5 1.2 4.2 0.1 0.0 0.0 4.5 91.2 100 100 100.00 38 217331800 25.9 23.7 23.7 26.6 0.0 37.1 21.4 smartseq 249418 SRR2049392 SRP059035 SRS951979 SRX1047472 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702640: LC-PT-45_DAPT; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702640 GSM1702640 LC-PT-45_DAPT 540981200 2704906 2015-06-05 16:18:03 310913832 540981200 2704906 2 2704906 index:0,count:2704906,average:100,stdev:0|index:1,count:2704906,average:100,stdev:0 GSM1702640_r1 0.89 2.68 0.04 504958560 690285483 480369833 663707406 136.7 138.17 2597555 1661361 398.242 3096.776 278 4950 91.48 96.23 2927352 2376293 2927352 2376293 90.67 91.66 2927352 2355315 2927352 2263343 8839539 1.75 1.02 0 4.74 0 0.06 0 0.01 0 0.00 0 3.90 0 2597555 0 200 0 197.28 0 1.53 0 0.01 0 1.52 0 0.01 0 170.84 0 0.29 0 27700 0 2704906 0 128225 0 1684 0 278 0 0 0 105389 0 568 0 0 0 11770 0 1486009 0 6207 0 1504554 0 91.29 0 2469330 0 96201 1537624 15.983451315475 2704906.0 2597555.0 27700.0 128225.0 1684.0 278.0 0.0 105389.0 2469330.0 96.0 1.0 4.7 0.1 0.0 0.0 3.9 91.3 100 100 100.00 38 270490600 25.7 24.0 24.0 26.3 0.0 37.2 21.6 smartseq 249422 SRR2049393 SRP059035 SRS951978 SRX1047473 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702641: LC-PT-45_Docetaxel; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702641 GSM1702641 LC-PT-45_Docetaxel 525141400 2625707 2015-06-05 16:18:03 302932430 525141400 2625707 2 2625707 index:0,count:2625707,average:100,stdev:0|index:1,count:2625707,average:100,stdev:0 GSM1702641_r1 1.14 2.69 0.04 485295182 658151796 459655983 632338723 135.62 137.57 2502789 1609350 393.257 3048.389 263 4786 90.34 95.46 2891538 2261136 2891538 2261136 89.65 90.89 2891538 2243730 2891538 2152860 10325022 2.13 1.08 0 5.10 0 0.08 0 0.01 0 0.00 0 4.59 0 2502789 0 200 0 197.22 0 1.54 0 0.01 0 1.52 0 0.01 0 145.42 0 0.29 0 28397 0 2625707 0 134042 0 2036 0 348 0 0 0 120534 0 622 0 0 0 10752 0 1429235 0 6236 0 1446845 0 90.21 0 2368747 0 100050 1477753 14.770144927536 2625707.0 2502789.0 28397.0 134042.0 2036.0 348.0 0.0 120534.0 2368747.0 95.3 1.1 5.1 0.1 0.0 0.0 4.6 90.2 100 100 100.00 38 262570700 25.8 23.9 24.0 26.4 0.0 37.1 21.5 smartseq 249426 SRR2049394 SRP059035 SRS951977 SRX1047474 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702642: LC-PT-45_Pooled; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702642 GSM1702642 LC-PT-45_Pooled 1731623790 8572395 2015-06-05 16:18:03 1187882512 1731623790 8572395 2 8572395 index:0,count:8572395,average:101,stdev:0|index:1,count:8572395,average:101,stdev:0 GSM1702642_r1 3.31 3.19 0.06 1499480642 2060356581 1442323122 1987879140 137.4 137.82 8323513 6577436 276.771 1770.842 176 31658 91.31 95.09 8849360 7600327 8849360 7600327 89.65 90.08 8849360 7462209 8849360 7200079 35017032 2.34 1.14 0 3.86 0 0.06 0 0.02 0 0.00 0 2.82 0 8323513 0 202 0 199.02 0 1.50 0 0.01 0 1.51 0 0.01 0 205.74 0 0.32 0 97540 0 8572395 0 330498 0 5298 0 1564 0 0 0 242020 0 2133 0 0 0 31095 0 4508607 0 16022 0 4557857 0 93.24 0 7993015 0 126880 4394155 34.632369167718 8572395.0 8323513.0 97540.0 330498.0 5298.0 1564.0 0.0 242020.0 7993015.0 97.1 1.1 3.9 0.1 0.0 0.0 2.8 93.2 101 101 101.00 38 865811895 26.8 23.2 23.2 26.7 0.0 36.4 23.3 smartseq 249430 SRR2049395 SRP059035 SRS951975 SRX1047475 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702643: LC-PT-45_Pt_tumor; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;primary tumor|source_name;;primary tumor, LC-PT-45 GEO Accession;;GSM1702643 GSM1702643 LC-PT-45_Pt_tumor 2283698476 11305438 2015-06-05 16:18:03 1565098345 2283698476 11305438 2 11305438 index:0,count:11305438,average:101,stdev:0|index:1,count:11305438,average:101,stdev:0 GSM1702643_r1 4.71 2.71 0.08 1832173515 2592530765 1736546954 2474934448 141.5 142.52 10345057 8732915 251.528 5402.909 176 49860 87.76 92.81 11359510 9078423 11359510 9078423 85.89 86.76 11359510 8885454 11359510 8486614 67977937 3.71 1.44 0 4.98 0 0.17 0 0.07 0 0.00 0 8.26 0 10345057 0 202 0 198.16 0 1.53 0 0.01 0 1.61 0 0.01 0 138.91 0 0.48 0 162822 0 11305438 0 563134 0 19545 0 7425 0 0 0 933411 0 1508 0 0 0 24707 0 4777167 0 23808 0 4827190 0 86.52 0 9781923 0 83785 4793980 57.217640389091 11305438.0 10345057.0 162822.0 563134.0 19545.0 7425.0 0.0 933411.0 9781923.0 91.5 1.4 5.0 0.2 0.1 0.0 8.3 86.5 101 101 101.00 38 1141849238 26.1 23.5 23.6 26.8 0.0 35.3 18.6 smartseq 249434 SRR2049396 SRP059035 SRS951976 SRX1047476 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702644: LC-PT-45_SC01; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702644 GSM1702644 LC-PT-45_SC01 1006099380 4980690 2015-06-05 16:18:03 681284187 1006099380 4980690 2 4980690 index:0,count:4980690,average:101,stdev:0|index:1,count:4980690,average:101,stdev:0 GSM1702644_r1 7.41 2.91 0.08 741334273 993664690 711688582 964792887 134.04 135.56 4171903 3816161 251.401 1499.829 160 20303 66.85 69.88 4627587 2789023 4627587 2789023 65.79 66.8 4627587 2744582 4627587 2666054 129145573 17.42 2.46 0 3.63 0 0.36 0 0.05 0 0.00 0 15.83 0 4171903 0 202 0 197.96 0 1.49 0 0.02 0 1.51 0 0.01 0 104.25 0 0.56 0 122580 0 4980690 0 180827 0 17880 0 2405 0 0 0 788502 0 621 0 0 0 7241 0 942367 0 9631 0 959860 0 80.13 0 3991076 0 46152 948666 20.555252210088 4980690.0 4171903.0 122580.0 180827.0 17880.0 2405.0 0.0 788502.0 3991076.0 83.8 2.5 3.6 0.4 0.0 0.0 15.8 80.1 101 101 101.00 38 503049690 28.0 21.2 20.9 29.9 0.0 34.6 16.1 smartseq 249438 SRR2049397 SRP059035 SRS951974 SRX1047477 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702645: LC-PT-45_SC02; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702645 GSM1702645 LC-PT-45_SC02 1785166314 8837457 2015-06-05 16:18:03 1208893049 1785166314 8837457 2 8837457 index:0,count:8837457,average:101,stdev:0|index:1,count:8837457,average:101,stdev:0 GSM1702645_r1 3.76 3.22 0.09 1378610449 1731748809 1322472251 1680835848 125.62 127.1 7686318 7100443 253.718 1055.646 176 35514 57.77 60.34 8601543 4440557 8601543 4440557 56.56 57.15 8601543 4347677 8601543 4205602 321043402 23.29 1.91 0 3.70 0 0.22 0 0.09 0 0.00 0 12.71 0 7686318 0 202 0 198.71 0 1.55 0 0.02 0 1.51 0 0.01 0 162.32 0 0.44 0 168904 0 8837457 0 327034 0 19704 0 7940 0 0 0 1123495 0 588 0 0 0 10437 0 1659998 0 16805 0 1687828 0 83.27 0 7359284 0 34928 1666508 47.712666055886 8837457.0 7686318.0 168904.0 327034.0 19704.0 7940.0 0.0 1123495.0 7359284.0 87.0 1.9 3.7 0.2 0.1 0.0 12.7 83.3 101 101 101.00 38 892583157 28.6 20.8 20.7 29.9 0.0 34.9 16.7 smartseq 249442 SRR2049398 SRP059035 SRS951973 SRX1047478 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702646: LC-PT-45_SC04; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702646 GSM1702646 LC-PT-45_SC04 1520279674 7526137 2015-06-05 16:18:03 1030953849 1520279674 7526137 2 7526137 index:0,count:7526137,average:101,stdev:0|index:1,count:7526137,average:101,stdev:0 GSM1702646_r1 3.77 2.69 0.1 1156348827 1478994214 1111185865 1430979828 127.9 128.78 6459855 5843437 257.671 1575.459 160 29477 61.36 64.02 7036310 3963867 7036310 3963867 60.29 60.86 7036310 3894387 7036310 3768380 254844001 22.04 2.26 0 3.56 0 0.22 0 0.04 0 0.00 0 13.90 0 6459855 0 202 0 198.31 0 1.51 0 0.02 0 1.52 0 0.01 0 128.41 0 0.56 0 169879 0 7526137 0 267779 0 16816 0 3035 0 0 0 1046431 0 802 0 0 0 12623 0 1607755 0 15372 0 1636552 0 82.27 0 6192076 0 59939 1595734 26.622633010227 7526137.0 6459855.0 169879.0 267779.0 16816.0 3035.0 0.0 1046431.0 6192076.0 85.8 2.3 3.6 0.2 0.0 0.0 13.9 82.3 101 101 101.00 38 760139837 28.2 21.3 21.0 29.6 0.0 34.9 16.6 smartseq 249446 SRR2049399 SRP059035 SRS951972 SRX1047479 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702647: LC-PT-45_SC05; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702647 GSM1702647 LC-PT-45_SC05 1899010080 9401040 2015-06-05 16:18:03 1295533781 1899010080 9401040 2 9401040 index:0,count:9401040,average:101,stdev:0|index:1,count:9401040,average:101,stdev:0 GSM1702647_r1 6.1 2.82 0.06 1515676008 2030619681 1454780404 1963722387 133.97 134.98 8370242 7367855 265.289 1177.615 165 36588 74.13 77.39 9138859 6204844 9138859 6204844 72.84 73.48 9138859 6097193 9138859 5891419 195465151 12.90 1.44 0 3.75 0 0.19 0 0.03 0 0.00 0 10.75 0 8370242 0 202 0 198.92 0 1.49 0 0.01 0 1.52 0 0.01 0 142.80 0 0.45 0 135388 0 9401040 0 352877 0 17530 0 2922 0 0 0 1010346 0 1625 0 0 0 18859 0 2733422 0 18975 0 2772881 0 85.28 0 8017365 0 49054 2727911 55.610368165695 9401040.0 8370242.0 135388.0 352877.0 17530.0 2922.0 0.0 1010346.0 8017365.0 89.0 1.4 3.8 0.2 0.0 0.0 10.7 85.3 101 101 101.00 38 949505040 28.0 21.5 21.4 29.1 0.0 35.0 17.1 smartseq 249858 SRR2049400 SRP059035 SRS951971 SRX1047480 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702648: LC-PT-45_SC06; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702648 GSM1702648 LC-PT-45_SC06 1456143260 7208630 2015-06-05 16:18:03 991577516 1456143260 7208630 2 7208630 index:0,count:7208630,average:101,stdev:0|index:1,count:7208630,average:101,stdev:0 GSM1702648_r1 4.91 2.98 0.04 1150852745 1581527126 1102633251 1526443652 137.42 138.44 6367226 5442769 264.336 1447.991 176 28083 83.5 87.36 6912549 5316809 6912549 5316809 82.0 82.79 6912549 5220896 6912549 5038211 76258856 6.63 1.56 0 3.90 0 0.23 0 0.02 0 0.00 0 11.42 0 6367226 0 202 0 198.69 0 1.48 0 0.01 0 1.54 0 0.01 0 141.04 0 0.45 0 112525 0 7208630 0 281448 0 16504 0 1599 0 0 0 823301 0 1236 0 0 0 17529 0 2441205 0 19319 0 2479289 0 84.42 0 6085778 0 52667 2423683 46.019006208821 7208630.0 6367226.0 112525.0 281448.0 16504.0 1599.0 0.0 823301.0 6085778.0 88.3 1.6 3.9 0.2 0.0 0.0 11.4 84.4 101 101 101.00 38 728071630 28.1 21.3 21.2 29.3 0.0 35.0 17.1 smartseq 249862 SRR2049401 SRP059035 SRS951970 SRX1047481 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702649: LC-PT-45_SC09; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702649 GSM1702649 LC-PT-45_SC09 1368350222 6774011 2015-06-05 16:18:03 933728314 1368350222 6774011 2 6774011 index:0,count:6774011,average:101,stdev:0|index:1,count:6774011,average:101,stdev:0 GSM1702649_r1 8.87 2.35 0.04 1065495428 1508602134 1025312427 1464736101 141.59 142.86 5926838 5132029 260.725 1404.155 165 29648 86.96 90.61 6481344 5154244 6481344 5154244 84.88 85.95 6481344 5030688 6481344 4889065 48631802 4.56 1.79 0 3.52 0 0.24 0 0.02 0 0.00 0 12.25 0 5926838 0 202 0 198.46 0 1.49 0 0.02 0 1.51 0 0.01 0 137.00 0 0.48 0 121306 0 6774011 0 238490 0 16093 0 1151 0 0 0 829929 0 1318 0 0 0 13418 0 2152980 0 15698 0 2183414 0 83.97 0 5688348 0 52123 2131983 40.902921934655 6774011.0 5926838.0 121306.0 238490.0 16093.0 1151.0 0.0 829929.0 5688348.0 87.5 1.8 3.5 0.2 0.0 0.0 12.3 84.0 101 101 101.00 38 684175111 27.6 21.7 21.6 29.0 0.0 34.8 16.8 smartseq 249867 SRR2049402 SRP059035 SRS951969 SRX1047482 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702650: LC-PT-45_SC10; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702650 GSM1702650 LC-PT-45_SC10 1957014380 9688190 2015-06-05 16:18:03 1331225241 1957014380 9688190 2 9688190 index:0,count:9688190,average:101,stdev:0|index:1,count:9688190,average:101,stdev:0 GSM1702650_r1 5.8 2.91 0.02 1541792517 2122273349 1478952776 2051569377 137.65 138.72 8599858 7616801 252.999 1172.189 174 39762 77.9 81.34 9348454 6699604 9348454 6699604 76.88 77.44 9348454 6611929 9348454 6378727 166873447 10.82 1.64 0 3.75 0 0.12 0 0.02 0 0.00 0 11.09 0 8599858 0 202 0 198.66 0 1.48 0 0.01 0 1.54 0 0.01 0 158.53 0 0.45 0 158931 0 9688190 0 363327 0 11935 0 1626 0 0 0 1074771 0 918 0 0 0 18911 0 2773049 0 16667 0 2809545 0 85.02 0 8236531 0 41085 2760762 67.196349032494 9688190.0 8599858.0 158931.0 363327.0 11935.0 1626.0 0.0 1074771.0 8236531.0 88.8 1.6 3.8 0.1 0.0 0.0 11.1 85.0 101 101 101.00 38 978507190 28.1 21.4 21.2 29.3 0.0 35.0 17.0 smartseq 249871 SRR2049403 SRP059035 SRS951968 SRX1047483 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702651: LC-PT-45_SC13; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702651 GSM1702651 LC-PT-45_SC13 1434682578 7102389 2015-06-05 16:18:03 974185199 1434682578 7102389 2 7102389 index:0,count:7102389,average:101,stdev:0|index:1,count:7102389,average:101,stdev:0 GSM1702651_r1 19.88 2.32 0.07 1061204082 1472291115 1016189903 1428653780 138.74 140.59 6021603 5802716 237.961 851.809 160 30953 56.94 59.63 6769872 3428715 6769872 3428715 56.71 57.28 6769872 3414841 6769872 3293648 243441477 22.94 2.44 0 3.83 0 0.40 0 0.26 0 0.00 0 14.55 0 6021603 0 202 0 198.39 0 1.45 0 0.02 0 1.48 0 0.01 0 131.12 0 0.46 0 172956 0 7102389 0 271787 0 28493 0 18750 0 0 0 1033543 0 336 0 0 0 7016 0 710784 0 14121 0 732257 0 80.96 0 5749816 0 20871 736107 35.269368980883 7102389.0 6021603.0 172956.0 271787.0 28493.0 18750.0 0.0 1033543.0 5749816.0 84.8 2.4 3.8 0.4 0.3 0.0 14.6 81.0 101 101 101.00 38 717341289 28.1 21.3 21.0 29.6 0.0 34.7 16.5 smartseq 249874 SRR2049404 SRP059035 SRS951967 SRX1047484 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702652: LC-PT-45_SC14; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702652 GSM1702652 LC-PT-45_SC14 1404836674 6954637 2015-06-05 16:18:03 952254755 1404836674 6954637 2 6954637 index:0,count:6954637,average:101,stdev:0|index:1,count:6954637,average:101,stdev:0 GSM1702652_r1 2.96 2.57 0.23 1099156604 1446170792 1058329239 1402490620 131.57 132.52 6161642 5447749 252.533 1186.581 175 28206 73.12 76.07 6694375 4505661 6694375 4505661 71.9 72.37 6694375 4430144 6694375 4286652 145039455 13.20 1.56 0 3.43 0 0.23 0 0.07 0 0.00 0 11.11 0 6161642 0 202 0 198.61 0 1.53 0 0.02 0 1.50 0 0.01 0 192.59 0 0.43 0 108490 0 6954637 0 238714 0 15848 0 4709 0 0 0 772438 0 1250 0 0 0 13306 0 2049437 0 14463 0 2078456 0 85.17 0 5922928 0 42203 2031559 48.137786413288 6954637.0 6161642.0 108490.0 238714.0 15848.0 4709.0 0.0 772438.0 5922928.0 88.6 1.6 3.4 0.2 0.1 0.0 11.1 85.2 101 101 101.00 38 702418337 28.1 21.5 21.3 29.1 0.0 35.1 17.3 smartseq 249878 SRR2049405 SRP059035 SRS951966 SRX1047485 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702653: LC-PT-45_SC15; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702653 GSM1702653 LC-PT-45_SC15 1384867560 6855780 2015-06-05 16:18:03 943357515 1384867560 6855780 2 6855780 index:0,count:6855780,average:101,stdev:0|index:1,count:6855780,average:101,stdev:0 GSM1702653_r1 17.47 1.8 0.07 1058525894 1506630804 1019991087 1463080864 142.33 143.44 5946277 5410216 247.645 1029.890 165 29467 76.52 79.63 6436328 4550294 6436328 4550294 75.55 76.23 6436328 4492125 6436328 4356181 126447394 11.95 1.82 0 3.38 0 0.27 0 0.02 0 0.00 0 12.98 0 5946277 0 202 0 198.53 0 1.45 0 0.02 0 1.45 0 0.01 0 119.23 0 0.47 0 124965 0 6855780 0 231769 0 18284 0 1612 0 0 0 889607 0 881 0 0 0 9677 0 1537820 0 13843 0 1562221 0 83.35 0 5714508 0 37704 1532130 40.635741565882 6855780.0 5946277.0 124965.0 231769.0 18284.0 1612.0 0.0 889607.0 5714508.0 86.7 1.8 3.4 0.3 0.0 0.0 13.0 83.4 101 101 101.00 38 692433780 28.1 21.4 21.1 29.4 0.0 34.7 16.5 smartseq 249882 SRR2049406 SRP059035 SRS951965 SRX1047486 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702654: LC-PT-45_SC23; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702654 GSM1702654 LC-PT-45_SC23 2202613656 10904028 2015-06-05 16:18:03 1502414010 2202613656 10904028 2 10904028 index:0,count:10904028,average:101,stdev:0|index:1,count:10904028,average:101,stdev:0 GSM1702654_r1 10.17 2.42 0.11 1716199705 2408320289 1646283029 2331491662 140.33 141.62 9574423 8569362 257.114 978.698 165 49680 75.4 78.78 10457900 7219327 10457900 7219327 74.15 74.82 10457900 7099816 10457900 6856134 209238184 12.19 1.75 0 3.76 0 0.20 0 0.02 0 0.00 0 11.97 0 9574423 0 202 0 198.66 0 1.42 0 0.01 0 1.46 0 0.01 0 141.71 0 0.46 0 190661 0 10904028 0 410375 0 22307 0 2353 0 0 0 1304945 0 1102 0 0 0 16343 0 2956412 0 21676 0 2995533 0 84.04 0 9164048 0 25129 3005721 119.611643917386 10904028.0 9574423.0 190661.0 410375.0 22307.0 2353.0 0.0 1304945.0 9164048.0 87.8 1.7 3.8 0.2 0.0 0.0 12.0 84.0 101 101 101.00 38 1101306828 27.8 21.6 21.4 29.1 0.0 34.8 17.0 smartseq 249886 SRR2049407 SRP059035 SRS951964 SRX1047487 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702655: LC-PT-45_SC28; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702655 GSM1702655 LC-PT-45_SC28 2201784446 10899923 2015-06-05 16:18:03 1493362997 2201784446 10899923 2 10899923 index:0,count:10899923,average:101,stdev:0|index:1,count:10899923,average:101,stdev:0 GSM1702655_r1 6.88 3.77 0.05 1728027047 2269409444 1659993570 2201340061 131.33 132.61 9628220 8833436 254.501 980.252 176 44549 63.56 66.28 10435488 6119396 10435488 6119396 63.2 63.59 10435488 6084648 10435488 5871040 353615982 20.46 1.67 0 3.63 0 0.19 0 0.04 0 0.00 0 11.44 0 9628220 0 202 0 198.88 0 1.51 0 0.02 0 1.47 0 0.01 0 155.10 0 0.43 0 181566 0 10899923 0 395847 0 20529 0 4750 0 0 0 1246424 0 902 0 0 0 12065 0 2239189 0 21166 0 2273322 0 84.70 0 9232373 0 34450 2269356 65.873904208999 10899923.0 9628220.0 181566.0 395847.0 20529.0 4750.0 0.0 1246424.0 9232373.0 88.3 1.7 3.6 0.2 0.0 0.0 11.4 84.7 101 101 101.00 38 1100892223 28.7 20.7 20.6 30.0 0.0 35.0 17.1 smartseq 249890 SRR2049408 SRP059035 SRS951963 SRX1047488 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702656: LC-PT-45_SC30; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702656 GSM1702656 LC-PT-45_SC30 2122996366 10509883 2015-06-05 16:18:03 1444481321 2122996366 10509883 2 10509883 index:0,count:10509883,average:101,stdev:0|index:1,count:10509883,average:101,stdev:0 GSM1702656_r1 10.99 2.8 0.04 1675250008 2312203585 1609349676 2235358120 138.02 138.9 9347245 8234947 255.088 1164.998 175 43458 78.76 82.17 10029489 7361458 10029489 7361458 77.66 78.15 10029489 7258976 10029489 7001208 174511345 10.42 1.51 0 3.70 0 0.17 0 0.02 0 0.00 0 10.87 0 9347245 0 202 0 198.80 0 1.49 0 0.01 0 1.47 0 0.01 0 141.71 0 0.44 0 158563 0 10509883 0 388748 0 18331 0 1799 0 0 0 1142508 0 2355 0 0 0 20412 0 3162089 0 21725 0 3206581 0 85.24 0 8958497 0 45888 3133029 68.275562238494 10509883.0 9347245.0 158563.0 388748.0 18331.0 1799.0 0.0 1142508.0 8958497.0 88.9 1.5 3.7 0.2 0.0 0.0 10.9 85.2 101 101 101.00 38 1061498183 28.2 21.3 21.2 29.3 0.0 35.0 17.2 smartseq 249894 SRR2049409 SRP059035 SRS951962 SRX1047489 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702657: LC-PT-45_SC34; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702657 GSM1702657 LC-PT-45_SC34 1976465566 9784483 2015-06-05 16:18:03 1342579477 1976465566 9784483 2 9784483 index:0,count:9784483,average:101,stdev:0|index:1,count:9784483,average:101,stdev:0 GSM1702657_r1 5.9 2.72 0.06 1554012308 1990154125 1493312991 1921483200 128.07 128.67 8658104 7860934 255.738 1115.798 175 40275 61.95 64.6 9268598 5363791 9268598 5363791 61.29 61.34 9268598 5306689 9268598 5093598 304639016 19.60 1.52 0 3.62 0 0.20 0 0.05 0 0.00 0 11.26 0 8658104 0 202 0 198.92 0 1.52 0 0.02 0 1.52 0 0.01 0 132.42 0 0.46 0 148935 0 9784483 0 354553 0 19160 0 5028 0 0 0 1102191 0 1128 0 0 0 17319 0 2268163 0 19520 0 2306130 0 84.86 0 8303551 0 46760 2266903 48.479533789564 9784483.0 8658104.0 148935.0 354553.0 19160.0 5028.0 0.0 1102191.0 8303551.0 88.5 1.5 3.6 0.2 0.1 0.0 11.3 84.9 101 101 101.00 38 988232783 28.4 21.2 21.0 29.5 0.0 35.0 17.1 smartseq 249922 SRR2049410 SRP059035 SRS951961 SRX1047490 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702658: LC-PT-45_SC35; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702658 GSM1702658 LC-PT-45_SC35 2290650912 11339856 2015-06-05 16:18:03 1560385452 2290650912 11339856 2 11339856 index:0,count:11339856,average:101,stdev:0|index:1,count:11339856,average:101,stdev:0 GSM1702658_r1 9.36 2.9 0.04 1822242707 2593301144 1750571785 2508166375 142.31 143.28 10154711 8725111 256.565 1254.604 175 47335 89.24 93.09 10984415 9061789 10984415 9061789 87.64 88.56 10984415 8899693 10984415 8621166 65945956 3.62 1.36 0 3.70 0 0.20 0 0.02 0 0.00 0 10.23 0 10154711 0 202 0 198.82 0 1.49 0 0.01 0 1.54 0 0.01 0 154.05 0 0.43 0 154218 0 11339856 0 420083 0 22354 0 2193 0 0 0 1160598 0 2166 0 0 0 29860 0 4074533 0 21748 0 4128307 0 85.84 0 9734628 0 47467 3996934 84.204478901131 11339856.0 10154711.0 154218.0 420083.0 22354.0 2193.0 0.0 1160598.0 9734628.0 89.5 1.4 3.7 0.2 0.0 0.0 10.2 85.8 101 101 101.00 38 1145325456 27.9 21.6 21.5 28.9 0.0 35.1 17.5 smartseq 249926 SRR2049411 SRP059035 SRS951959 SRX1047491 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702659: LC-PT-45_SC45; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702659 GSM1702659 LC-PT-45_SC45 1617391780 8006890 2015-06-05 16:18:03 1101062854 1617391780 8006890 2 8006890 index:0,count:8006890,average:101,stdev:0|index:1,count:8006890,average:101,stdev:0 GSM1702659_r1 5.18 3.04 0.07 1271608146 1683557544 1223968242 1628847131 132.4 133.08 7105740 6291459 255.471 1243.146 176 32928 71.69 74.66 7616012 5093877 7616012 5093877 70.44 70.86 7616012 5005027 7616012 4834768 184549866 14.51 1.59 0 3.53 0 0.22 0 0.04 0 0.00 0 11.00 0 7105740 0 202 0 198.74 0 1.53 0 0.02 0 1.52 0 0.01 0 134.07 0 0.47 0 127703 0 8006890 0 282877 0 17449 0 2865 0 0 0 880836 0 1177 0 0 0 16560 0 2334271 0 17687 0 2369695 0 85.21 0 6822863 0 52349 2302973 43.992683718887 8006890.0 7105740.0 127703.0 282877.0 17449.0 2865.0 0.0 880836.0 6822863.0 88.7 1.6 3.5 0.2 0.0 0.0 11.0 85.2 101 101 101.00 38 808695890 28.1 21.5 21.2 29.2 0.0 35.0 17.3 smartseq 249930 SRR2049412 SRP059035 SRS951958 SRX1047492 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702660: LC-PT-45_SC49; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702660 GSM1702660 LC-PT-45_SC49 2536128180 12555090 2015-06-05 16:18:03 1724887402 2536128180 12555090 2 12555090 index:0,count:12555090,average:101,stdev:0|index:1,count:12555090,average:101,stdev:0 GSM1702660_r1 6.42 2.7 0.09 1963171266 2647453635 1884104942 2556399607 134.86 135.68 11171141 9966192 242.823 1076.344 165 55505 74.49 77.82 12112818 8321530 12112818 8321530 73.09 73.64 12112818 8165471 12112818 7874611 243273024 12.39 1.43 0 3.81 0 0.22 0 0.02 0 0.00 0 10.79 0 11171141 0 202 0 198.67 0 1.48 0 0.01 0 1.49 0 0.01 0 159.15 0 0.45 0 180092 0 12555090 0 477789 0 27091 0 2403 0 0 0 1354455 0 1891 0 0 0 27418 0 3848350 0 26168 0 3903827 0 85.17 0 10693352 0 54929 3753126 68.326858307998 12555090.0 11171141.0 180092.0 477789.0 27091.0 2403.0 0.0 1354455.0 10693352.0 89.0 1.4 3.8 0.2 0.0 0.0 10.8 85.2 101 101 101.00 38 1268064090 27.8 21.8 21.5 28.8 0.0 35.0 17.3 smartseq 249951 SRR2049417 SRP059035 SRS951954 SRX1047497 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702665: LC-PT-45_SC61; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702665 GSM1702665 LC-PT-45_SC61 2336492186 11566793 2015-06-05 16:18:03 1583499531 2336492186 11566793 2 11566793 index:0,count:11566793,average:101,stdev:0|index:1,count:11566793,average:101,stdev:0 GSM1702665_r1 3.72 2.77 0.07 1793938169 2299889278 1734987365 2241145764 128.2 129.17 10171977 9328286 241.254 1038.105 175 50265 62.36 64.6 10927496 6343085 10927496 6343085 61.38 61.76 10927496 6243802 10927496 6064824 376476177 20.99 1.46 0 3.05 0 0.24 0 0.08 0 0.00 0 11.74 0 10171977 0 202 0 198.89 0 1.51 0 0.02 0 1.53 0 0.01 0 120.70 0 0.43 0 168756 0 11566793 0 352230 0 27337 0 9087 0 0 0 1358392 0 1258 0 0 0 18321 0 2517280 0 18047 0 2554906 0 84.90 0 9819747 0 42220 2489919 58.974869729986 11566793.0 10171977.0 168756.0 352230.0 27337.0 9087.0 0.0 1358392.0 9819747.0 87.9 1.5 3.0 0.2 0.1 0.0 11.7 84.9 101 101 101.00 38 1168246093 28.7 20.9 20.6 29.8 0.0 35.0 17.1 smartseq 249955 SRR2049418 SRP059035 SRS951953 SRX1047498 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702666: LC-PT-45_SC65; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702666 GSM1702666 LC-PT-45_SC65 1632521378 8081789 2015-06-05 16:18:03 1095858782 1632521378 8081789 2 8081789 index:0,count:8081789,average:101,stdev:0|index:1,count:8081789,average:101,stdev:0 GSM1702666_r1 17.7 1.96 0.18 1108586130 1499546794 1045261937 1443348435 135.27 138.08 6460658 6140563 228.380 867.985 155 33995 56.83 60.66 7488245 3671474 7488245 3671474 57.03 58.3 7488245 3684523 7488245 3528949 248850471 22.45 2.42 0 5.05 0 0.75 0 0.13 0 0.00 0 19.18 0 6460658 0 202 0 197.57 0 1.47 0 0.02 0 1.51 0 0.01 0 72.37 0 0.50 0 195664 0 8081789 0 407778 0 60904 0 10448 0 0 0 1549779 0 527 0 0 0 8399 0 1009293 0 15670 0 1033889 0 74.90 0 6052880 0 24473 1094204 44.710660728149 8081789.0 6460658.0 195664.0 407778.0 60904.0 10448.0 0.0 1549779.0 6052880.0 79.9 2.4 5.0 0.8 0.1 0.0 19.2 74.9 101 101 101.00 38 816260689 27.8 21.2 21.0 30.0 0.0 34.3 15.6 smartseq 249959 SRR2049419 SRP059035 SRS951952 SRX1047499 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702667: LC-PT-45_SC67; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702667 GSM1702667 LC-PT-45_SC67 2140419472 10596136 2015-06-05 16:18:03 1452464303 2140419472 10596136 2 10596136 index:0,count:10596136,average:101,stdev:0|index:1,count:10596136,average:101,stdev:0 GSM1702667_r1 3.13 2.66 0.09 1618586146 2041846154 1562773976 1986254952 126.15 127.1 9256554 8528852 240.250 1252.744 165 47090 60.03 62.34 9994015 5557094 9994015 5557094 58.97 59.35 9994015 5458603 9994015 5289979 345929306 21.37 1.96 0 3.24 0 0.26 0 0.06 0 0.00 0 12.32 0 9256554 0 202 0 198.44 0 1.53 0 0.02 0 1.48 0 0.01 0 126.31 0 0.51 0 208138 0 10596136 0 342952 0 27825 0 6424 0 0 0 1305333 0 764 0 0 0 16386 0 2098000 0 21652 0 2136802 0 84.12 0 8913602 0 53854 2072816 38.489545809039 10596136.0 9256554.0 208138.0 342952.0 27825.0 6424.0 0.0 1305333.0 8913602.0 87.4 2.0 3.2 0.3 0.1 0.0 12.3 84.1 101 101 101.00 38 1070209736 28.3 21.3 20.9 29.5 0.0 35.0 17.1 smartseq 250059 SRR2049432 SRP059035 SRS951938 SRX1047512 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702680: LC-PT-45-Re_Carboplatin; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702680 GSM1702680 LC-PT-45-Re_Carboplatin 2165985400 10829927 2015-06-05 16:18:03 1424453766 2165985400 10829927 2 10829927 index:0,count:10829927,average:100,stdev:0|index:1,count:10829927,average:100,stdev:0 GSM1702680_r1 2.93 2.83 0.05 1790931805 2413940428 1714291185 2328169976 134.79 135.81 9886741 7312027 325.998 2598.931 165 25886 87.65 91.77 10893973 8665262 10893973 8665262 86.51 87.36 10893973 8552616 10893973 8248661 71729267 4.01 1.71 0 4.10 0 0.10 0 0.02 0 0.00 0 8.59 0 9886741 0 200 0 196.53 0 1.56 0 0.01 0 1.49 0 0.01 0 178.84 0 0.40 0 185251 0 10829927 0 444273 0 10890 0 2169 0 0 0 930127 0 2067 0 0 0 32877 0 4676351 0 24108 0 4735403 0 87.19 0 9442468 0 144882 4633069 31.978223657873 10829927.0 9886741.0 185251.0 444273.0 10890.0 2169.0 0.0 930127.0 9442468.0 91.3 1.7 4.1 0.1 0.0 0.0 8.6 87.2 100 100 100.00 38 1082992700 26.6 23.0 22.8 27.6 0.0 35.9 19.7 smartseq 250063 SRR2049433 SRP059035 SRS951939 SRX1047513 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702681: LC-PT-45-Re_DAPT; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702681 GSM1702681 LC-PT-45-Re_DAPT 1874084800 9370424 2015-06-05 16:18:03 1233257673 1874084800 9370424 2 9370424 index:0,count:9370424,average:100,stdev:0|index:1,count:9370424,average:100,stdev:0 GSM1702681_r1 2.71 2.8 0.05 1543421784 2092468293 1474338499 2014585052 135.57 136.64 8505280 6248343 329.851 2705.646 165 22292 88.89 93.29 9438924 7560559 9438924 7560559 87.89 88.84 9438924 7475470 9438924 7199802 47749295 3.09 1.84 0 4.28 0 0.10 0 0.02 0 0.00 0 9.11 0 8505280 0 200 0 196.46 0 1.55 0 0.01 0 1.50 0 0.01 0 152.64 0 0.41 0 172295 0 9370424 0 400717 0 9814 0 1544 0 0 0 853786 0 1810 0 0 0 28150 0 4046455 0 21702 0 4098117 0 86.49 0 8104563 0 136576 4020966 29.441234184630 9370424.0 8505280.0 172295.0 400717.0 9814.0 1544.0 0.0 853786.0 8104563.0 90.8 1.8 4.3 0.1 0.0 0.0 9.1 86.5 100 100 100.00 38 937042400 26.4 23.1 22.9 27.6 0.0 35.9 19.5 smartseq 250066 SRR2049434 SRP059035 SRS951937 SRX1047514 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702682: LC-PT-45-Re_Docetaxel; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702682 GSM1702682 LC-PT-45-Re_Docetaxel 1501279800 7506399 2015-06-05 16:18:03 997363855 1501279800 7506399 2 7506399 index:0,count:7506399,average:100,stdev:0|index:1,count:7506399,average:100,stdev:0 GSM1702682_r1 2.91 2.73 0.05 1217803321 1647760838 1157643484 1582098659 135.31 136.67 6740804 4967829 322.864 2814.221 159 18112 87.67 92.47 7602937 5909795 7602937 5909795 86.72 87.8 7602937 5845320 7602937 5611779 42659650 3.50 1.93 0 4.66 0 0.11 0 0.02 0 0.00 0 10.07 0 6740804 0 200 0 196.20 0 1.54 0 0.01 0 1.52 0 0.01 0 153.54 0 0.46 0 144775 0 7506399 0 349541 0 8095 0 1424 0 0 0 756076 0 1381 0 0 0 23181 0 3239158 0 17943 0 3281663 0 85.14 0 6391263 0 134730 3217610 23.881911972092 7506399.0 6740804.0 144775.0 349541.0 8095.0 1424.0 0.0 756076.0 6391263.0 89.8 1.9 4.7 0.1 0.0 0.0 10.1 85.1 100 100 100.00 38 750639900 26.3 23.2 23.1 27.4 0.0 35.9 19.5 smartseq 250070 SRR2049435 SRP059035 SRS951936 SRX1047515 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702683: LC-PT-45-Re_Selumetinib; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702683 GSM1702683 LC-PT-45-Re_Selumetinib 1413644400 7068222 2015-06-05 16:18:03 936299912 1413644400 7068222 2 7068222 index:0,count:7068222,average:100,stdev:0|index:1,count:7068222,average:100,stdev:0 GSM1702683_r1 3.32 2.78 0.05 1152134921 1561360842 1102268672 1504046035 135.52 136.45 6385016 4753146 321.486 2741.122 165 17279 87.46 91.63 7039113 5584638 7039113 5584638 86.2 87.02 7039113 5503652 7039113 5303420 46569389 4.04 2.09 0 4.11 0 0.11 0 0.02 0 0.00 0 9.54 0 6385016 0 200 0 196.27 0 1.55 0 0.01 0 1.50 0 0.01 0 142.95 0 0.44 0 147553 0 7068222 0 290468 0 7597 0 1560 0 0 0 674049 0 1281 0 0 0 21583 0 2956492 0 16883 0 2996239 0 86.22 0 6094548 0 127704 2933501 22.971097224832 7068222.0 6385016.0 147553.0 290468.0 7597.0 1560.0 0.0 674049.0 6094548.0 90.3 2.1 4.1 0.1 0.0 0.0 9.5 86.2 100 100 100.00 38 706822200 26.4 23.2 23.0 27.4 0.0 35.9 19.7 smartseq 250074 SRR2049436 SRP059035 SRS951933 SRX1047516 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702684: LC-PT-45-Re_Pooled; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702684 GSM1702684 LC-PT-45-Re_Pooled 2007413800 10037069 2015-06-05 16:18:03 1423001495 2007413800 10037069 2 10037069 index:0,count:10037069,average:100,stdev:0|index:1,count:10037069,average:100,stdev:0 GSM1702684_r1 1.45 2.49 0.05 1441509127 1944711593 1346765424 1847669737 134.91 137.19 8569725 6985686 254.391 2070.880 159 32600 89.07 95.72 10248031 7632766 10248031 7632766 88.27 90.22 10248031 7564876 10248031 7194739 25851093 1.79 1.76 0 5.93 0 0.13 0 0.01 0 0.00 0 14.48 0 8569725 0 200 0 194.86 0 1.51 0 0.01 0 1.48 0 0.01 0 118.47 0 0.56 0 176268 0 10037069 0 595307 0 12670 0 887 0 0 0 1453787 0 1755 0 0 0 27434 0 4186170 0 21196 0 4236555 0 79.45 0 7974418 0 127298 3968904 31.178054643435 10037069.0 8569725.0 176268.0 595307.0 12670.0 887.0 0.0 1453787.0 7974418.0 85.4 1.8 5.9 0.1 0.0 0.0 14.5 79.4 100 100 100.00 38 1003706900 25.9 23.6 23.0 27.5 0.0 34.8 18.3 smartseq 250079 SRR2049437 SRP059035 SRS951934 SRX1047517 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702685: LC-PT-45-Re_SC01; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702685 GSM1702685 LC-PT-45-Re_SC01 1280506200 6402531 2015-06-05 16:18:03 831153841 1280506200 6402531 2 6402531 index:0,count:6402531,average:100,stdev:0|index:1,count:6402531,average:100,stdev:0 GSM1702685_r1 3.96 2.35 0.08 969804992 1246434993 934054086 1209961452 128.52 129.54 5669914 5062295 245.150 1365.640 143 25762 65.23 67.86 6149020 3698214 6149020 3698214 63.64 64.05 6149020 3608220 6149020 3490746 169410135 17.47 1.61 0 3.44 0 0.16 0 0.12 0 0.00 0 11.15 0 5669914 0 200 0 196.48 0 1.55 0 0.02 0 1.54 0 0.01 0 136.39 0 0.33 0 103022 0 6402531 0 220297 0 10526 0 7899 0 0 0 714192 0 674 0 0 0 13285 0 1656417 0 13608 0 1683984 0 85.12 0 5449617 0 42105 1589345 37.747179669873 6402531.0 5669914.0 103022.0 220297.0 10526.0 7899.0 0.0 714192.0 5449617.0 88.6 1.6 3.4 0.2 0.1 0.0 11.2 85.1 100 100 100.00 38 640253100 27.4 21.9 21.5 29.1 0.0 35.9 18.3 smartseq 250083 SRR2049438 SRP059035 SRS951932 SRX1047518 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702686: LC-PT-45-Re_SC06; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702686 GSM1702686 LC-PT-45-Re_SC06 1280567000 6402835 2015-06-05 16:18:03 837151987 1280567000 6402835 2 6402835 index:0,count:6402835,average:100,stdev:0|index:1,count:6402835,average:100,stdev:0 GSM1702686_r1 3.44 3.12 0.05 1084718813 1390853398 1049512707 1352119618 128.22 128.83 5888180 5095625 287.451 1404.625 185 24138 67.74 70.12 6297137 3988933 6297137 3988933 66.41 66.71 6297137 3910555 6297137 3794597 182026404 16.78 1.49 0 3.12 0 0.14 0 0.03 0 0.00 0 7.87 0 5888180 0 200 0 197.46 0 1.57 0 0.01 0 1.53 0 0.01 0 140.55 0 0.34 0 95126 0 6402835 0 199762 0 9233 0 1686 0 0 0 503736 0 1067 0 0 0 12646 0 1844225 0 14579 0 1872517 0 88.84 0 5688418 0 44862 1862949 41.526213722081 6402835.0 5888180.0 95126.0 199762.0 9233.0 1686.0 0.0 503736.0 5688418.0 92.0 1.5 3.1 0.1 0.0 0.0 7.9 88.8 100 100 100.00 38 640283500 27.6 21.6 21.6 29.2 0.0 35.9 18.4 smartseq 250311 SRR2049471 SRP059035 SRS951900 SRX1047551 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702719: LC-PT-45-Re_SC78; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702719 GSM1702719 LC-PT-45-Re_SC78 1531596000 7657980 2015-06-05 16:18:03 995171209 1531596000 7657980 2 7657980 index:0,count:7657980,average:100,stdev:0|index:1,count:7657980,average:100,stdev:0 GSM1702719_r1 4.7 2.79 0.06 1180493995 1626304818 1134636679 1573760892 137.76 138.7 6855238 5731602 252.038 1508.977 163 29694 88.41 92.21 7429075 6060540 7429075 6060540 86.12 87.01 7429075 5903678 7429075 5719315 44557924 3.77 1.45 0 3.69 0 0.16 0 0.03 0 0.00 0 10.29 0 6855238 0 200 0 196.32 0 1.50 0 0.01 0 1.50 0 0.01 0 155.76 0 0.32 0 110694 0 7657980 0 282445 0 12553 0 2013 0 0 0 788176 0 1666 0 0 0 19364 0 3078415 0 16786 0 3116231 0 85.83 0 6572793 0 43732 2938332 67.189517973109 7657980.0 6855238.0 110694.0 282445.0 12553.0 2013.0 0.0 788176.0 6572793.0 89.5 1.4 3.7 0.2 0.0 0.0 10.3 85.8 100 100 100.00 38 765798000 27.0 22.4 22.2 28.5 0.0 35.9 18.8 smartseq 250319 SRR2049473 SRP059035 SRS951898 SRX1047553 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702721: LC-PT-45-Re_SC84; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702721 GSM1702721 LC-PT-45-Re_SC84 1250813200 6254066 2015-06-05 16:18:03 812077225 1250813200 6254066 2 6254066 index:0,count:6254066,average:100,stdev:0|index:1,count:6254066,average:100,stdev:0 GSM1702721_r1 2.93 2.54 0.11 1063022300 1356557439 1027574389 1318696284 127.61 128.33 5773812 4994837 287.721 1370.683 188 23413 68.66 71.14 6168009 3964098 6168009 3964098 67.39 67.79 6168009 3891056 6168009 3777159 178977322 16.84 1.57 0 3.23 0 0.15 0 0.04 0 0.00 0 7.48 0 5773812 0 200 0 197.38 0 1.55 0 0.01 0 1.53 0 0.01 0 156.35 0 0.33 0 98086 0 6254066 0 201764 0 9644 0 2724 0 0 0 467886 0 992 0 0 0 12653 0 1805233 0 15202 0 1834080 0 89.09 0 5572048 0 48824 1817036 37.216041291168 6254066.0 5773812.0 98086.0 201764.0 9644.0 2724.0 0.0 467886.0 5572048.0 92.3 1.6 3.2 0.2 0.0 0.0 7.5 89.1 100 100 100.00 38 625406600 27.9 21.3 21.3 29.5 0.0 36.0 18.7 smartseq 250323 SRR2049474 SRP059035 SRS951897 SRX1047554 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702722: LC-PT-45-Re_SC85; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702722 GSM1702722 LC-PT-45-Re_SC85 1329526800 6647634 2015-06-05 16:18:03 864277265 1329526800 6647634 2 6647634 index:0,count:6647634,average:100,stdev:0|index:1,count:6647634,average:100,stdev:0 GSM1702722_r1 3.57 2.71 0.02 987644871 1254768756 954281835 1217991324 127.05 127.63 5883422 5257562 242.862 1195.936 143 26642 66.32 68.82 6280079 3901690 6280079 3901690 65.18 65.45 6280079 3834642 6280079 3710579 174831894 17.70 1.82 0 3.22 0 0.14 0 0.04 0 0.00 0 11.31 0 5883422 0 200 0 195.96 0 1.55 0 0.01 0 1.51 0 0.01 0 147.73 0 0.35 0 120668 0 6647634 0 213748 0 9317 0 2763 0 0 0 752132 0 938 0 0 0 12739 0 1726034 0 15007 0 1754718 0 85.29 0 5669674 0 50338 1635153 32.483471731098 6647634.0 5883422.0 120668.0 213748.0 9317.0 2763.0 0.0 752132.0 5669674.0 88.5 1.8 3.2 0.1 0.0 0.0 11.3 85.3 100 100 100.00 38 664763400 27.8 21.6 21.2 29.4 0.0 36.0 18.8 smartseq 250327 SRR2049475 SRP059035 SRS951894 SRX1047555 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702723: LC-PT-45-Re_SC87; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702723 GSM1702723 LC-PT-45-Re_SC87 1246859600 6234298 2015-06-05 16:18:03 818829546 1246859600 6234298 2 6234298 index:0,count:6234298,average:100,stdev:0|index:1,count:6234298,average:100,stdev:0 GSM1702723_r1 2.86 2.8 0.06 945080716 1218517417 910473621 1180900657 128.93 129.7 5546059 4948762 241.932 1158.183 153 25586 66.92 69.6 5958091 3711202 5958091 3711202 65.66 65.95 5958091 3641812 5958091 3516462 161536940 17.09 1.63 0 3.43 0 0.15 0 0.04 0 0.00 0 10.84 0 5546059 0 200 0 196.27 0 1.59 0 0.02 0 1.51 0 0.01 0 147.65 0 0.37 0 101854 0 6234298 0 213883 0 9642 0 2599 0 0 0 675998 0 613 0 0 0 12089 0 1667018 0 13964 0 1693684 0 85.53 0 5332176 0 37930 1601414 42.220247824941 6234298.0 5546059.0 101854.0 213883.0 9642.0 2599.0 0.0 675998.0 5332176.0 89.0 1.6 3.4 0.2 0.0 0.0 10.8 85.5 100 100 100.00 38 623429800 27.7 21.7 21.4 29.2 0.0 36.0 18.7 smartseq 250331 SRR2049476 SRP059035 SRS951895 SRX1047556 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702724: LC-PT-45-Re_SC89; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702724 GSM1702724 LC-PT-45-Re_SC89 1354349200 6771746 2015-06-05 16:18:03 872962502 1354349200 6771746 2 6771746 index:0,count:6771746,average:100,stdev:0|index:1,count:6771746,average:100,stdev:0 GSM1702724_r1 3.29 2.8 0.1 1039531594 1287849742 1004358961 1250027242 123.89 124.46 6056470 5447402 251.449 1134.720 153 27254 59.77 62.01 6472737 3620117 6472737 3620117 58.66 58.8 6472737 3552792 6472737 3432584 241715943 23.25 1.57 0 3.22 0 0.15 0 0.06 0 0.00 0 10.35 0 6056470 0 200 0 196.59 0 1.53 0 0.02 0 1.49 0 0.01 0 171.68 0 0.34 0 106552 0 6771746 0 218347 0 10455 0 3780 0 0 0 701041 0 996 0 0 0 11363 0 1601502 0 15055 0 1628916 0 86.21 0 5838123 0 43876 1541444 35.131826055247 6771746.0 6056470.0 106552.0 218347.0 10455.0 3780.0 0.0 701041.0 5838123.0 89.4 1.6 3.2 0.2 0.1 0.0 10.4 86.2 100 100 100.00 38 677174600 27.9 21.5 21.0 29.6 0.0 36.0 18.6 smartseq 250335 SRR2049477 SRP059035 SRS951893 SRX1047557 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702725: LC-PT-45-Re_SC90; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702725 GSM1702725 LC-PT-45-Re_SC90 1422100200 7110501 2015-06-05 16:18:03 917066360 1422100200 7110501 2 7110501 index:0,count:7110501,average:100,stdev:0|index:1,count:7110501,average:100,stdev:0 GSM1702725_r1 3.35 2.88 0.08 1215252107 1510441360 1178678072 1471298520 124.29 124.83 6596614 5807593 287.345 1341.160 176 26941 60.83 62.79 6996274 4012398 6996274 4012398 59.79 59.95 6996274 3943939 6996274 3830704 253236960 20.84 1.64 0 2.91 0 0.11 0 0.04 0 0.00 0 7.07 0 6596614 0 200 0 197.56 0 1.55 0 0.02 0 1.54 0 0.01 0 192.46 0 0.33 0 116756 0 7110501 0 206631 0 8015 0 2850 0 0 0 503022 0 1240 0 0 0 13573 0 1770764 0 16141 0 1801718 0 89.87 0 6389983 0 46994 1793133 38.156637017492 7110501.0 6596614.0 116756.0 206631.0 8015.0 2850.0 0.0 503022.0 6389983.0 92.8 1.6 2.9 0.1 0.0 0.0 7.1 89.9 100 100 100.00 38 711050100 28.0 21.4 21.3 29.3 0.0 36.2 19.1 smartseq 250403 SRR2049488 SRP059035 SRS951883 SRX1047568 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702736: LC-MBT-15_SC07; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702736 GSM1702736 LC-MBT-15_SC07 1352789400 6763947 2015-06-05 16:18:03 896439151 1352789400 6763947 2 6763947 index:0,count:6763947,average:100,stdev:0|index:1,count:6763947,average:100,stdev:0 GSM1702736_r1 2.68 4.16 0.02 1033063981 1330310856 998015505 1289743249 128.77 129.23 6067487 5323272 245.747 1374.141 143 27373 70.06 72.7 6470551 4250804 6470551 4250804 68.47 68.67 6470551 4154209 6470551 4014892 149049076 14.43 1.53 0 3.26 0 0.09 0 0.03 0 0.00 0 10.17 0 6067487 0 200 0 196.27 0 1.57 0 0.02 0 1.48 0 0.01 0 135.28 0 0.40 0 103529 0 6763947 0 220669 0 6420 0 1833 0 0 0 688207 0 996 0 0 0 15604 0 1960664 0 14585 0 1991849 0 86.44 0 5846818 0 55073 1876269 34.068763277831 6763947.0 6067487.0 103529.0 220669.0 6420.0 1833.0 0.0 688207.0 5846818.0 89.7 1.5 3.3 0.1 0.0 0.0 10.2 86.4 100 100 100.00 38 676394700 27.3 22.0 21.5 29.1 0.0 35.7 18.3 smartseq 250407 SRR2049489 SRP059035 SRS951882 SRX1047569 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702737: LC-MBT-15_SC08; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702737 GSM1702737 LC-MBT-15_SC08 1202605600 6013028 2015-06-05 16:18:03 789450200 1202605600 6013028 2 6013028 index:0,count:6013028,average:100,stdev:0|index:1,count:6013028,average:100,stdev:0 GSM1702737_r1 2.17 4.01 0.02 802315941 1006692414 772718378 973763828 125.47 126.02 4988757 4453491 225.772 1498.052 111 24559 65.51 68.3 5357363 3268286 5357363 3268286 63.95 64.32 5357363 3190256 5357363 3077703 135983506 16.95 1.81 0 3.39 0 0.13 0 0.03 0 0.00 0 16.87 0 4988757 0 200 0 194.34 0 1.55 0 0.02 0 1.49 0 0.01 0 137.01 0 0.44 0 108797 0 6013028 0 203831 0 7820 0 2086 0 0 0 1014365 0 643 0 0 0 11851 0 1458588 0 13086 0 1484168 0 79.58 0 4784926 0 57434 1350663 23.516784483059 6013028.0 4988757.0 108797.0 203831.0 7820.0 2086.0 0.0 1014365.0 4784926.0 83.0 1.8 3.4 0.1 0.0 0.0 16.9 79.6 100 100 100.00 38 601302800 27.0 22.6 21.4 29.1 0.0 35.7 18.1 smartseq 250435 SRR2049490 SRP059035 SRS951880 SRX1047570 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702738: LC-MBT-15_SC09; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702738 GSM1702738 LC-MBT-15_SC09 1230708600 6153543 2015-06-05 16:18:03 808732332 1230708600 6153543 2 6153543 index:0,count:6153543,average:100,stdev:0|index:1,count:6153543,average:100,stdev:0 GSM1702738_r1 2.82 4.26 0.02 837797691 1058065263 809459859 1025292120 126.29 126.66 5178833 4688482 226.288 1220.167 111 25199 65.12 67.62 5518097 3372366 5518097 3372366 63.73 63.94 5518097 3300566 5518097 3188810 150388374 17.95 1.78 0 3.11 0 0.12 0 0.03 0 0.00 0 15.69 0 5178833 0 200 0 194.59 0 1.57 0 0.02 0 1.45 0 0.01 0 142.92 0 0.41 0 109522 0 6153543 0 191589 0 7498 0 2007 0 0 0 965205 0 699 0 0 0 10631 0 1373249 0 12091 0 1396670 0 81.05 0 4987244 0 48254 1276291 26.449434243793 6153543.0 5178833.0 109522.0 191589.0 7498.0 2007.0 0.0 965205.0 4987244.0 84.2 1.8 3.1 0.1 0.0 0.0 15.7 81.0 100 100 100.00 38 615354300 27.5 22.1 21.0 29.4 0.0 35.8 18.4 smartseq 250439 SRR2049491 SRP059035 SRS951879 SRX1047571 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702739: LC-MBT-15_SC10; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702739 GSM1702739 LC-MBT-15_SC10 628004800 3140024 2015-06-05 16:18:03 413966451 628004800 3140024 2 3140024 index:0,count:3140024,average:100,stdev:0|index:1,count:3140024,average:100,stdev:0 GSM1702739_r1 2.32 3.97 0.02 453080992 565239043 435039545 545372565 124.75 125.36 2655599 2391917 245.330 2034.077 160 12986 59.63 62.36 2864260 1583507 2864260 1583507 58.59 58.88 2864260 1555810 2864260 1495254 90612563 20.00 3.35 0 3.70 0 0.15 0 0.04 0 0.00 0 15.24 0 2655599 0 200 0 195.09 0 1.56 0 0.02 0 1.50 0 0.01 0 116.54 0 0.63 0 105221 0 3140024 0 116214 0 4593 0 1210 0 0 0 478622 0 265 0 0 0 6131 0 637928 0 7439 0 651763 0 80.87 0 2539385 0 57646 620293 10.760382333553 3140024.0 2655599.0 105221.0 116214.0 4593.0 1210.0 0.0 478622.0 2539385.0 84.6 3.4 3.7 0.1 0.0 0.0 15.2 80.9 100 100 100.00 38 314002400 26.7 22.3 21.5 29.5 0.0 35.3 17.1 smartseq 250443 SRR2049492 SRP059035 SRS951881 SRX1047572 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702740: LC-MBT-15_SC11; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702740 GSM1702740 LC-MBT-15_SC11 1494252800 7471264 2015-06-05 16:18:03 1005366566 1494252800 7471264 2 7471264 index:0,count:7471264,average:100,stdev:0|index:1,count:7471264,average:100,stdev:0 GSM1702740_r1 2.73 4.61 0.02 993254855 1225311764 961403539 1189082831 123.36 123.68 6179904 5631436 223.890 1273.923 111 31102 58.22 60.35 6576964 3598198 6576964 3598198 56.97 57.11 6576964 3520868 6576964 3405105 215804631 21.73 1.67 0 2.92 0 0.13 0 0.04 0 0.00 0 17.12 0 6179904 0 200 0 194.44 0 1.56 0 0.02 0 1.50 0 0.01 0 137.93 0 0.45 0 124814 0 7471264 0 217935 0 9365 0 2987 0 0 0 1279008 0 636 0 0 0 13141 0 1490965 0 16362 0 1521104 0 79.80 0 5961969 0 50153 1383846 27.592486989811 7471264.0 6179904.0 124814.0 217935.0 9365.0 2987.0 0.0 1279008.0 5961969.0 82.7 1.7 2.9 0.1 0.0 0.0 17.1 79.8 100 100 100.00 38 747126400 27.4 22.3 21.1 29.2 0.0 35.7 18.2 smartseq 250447 SRR2049493 SRP059035 SRS951878 SRX1047573 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702741: LC-MBT-15_SC14; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702741 GSM1702741 LC-MBT-15_SC14 1072270800 5361354 2015-06-05 16:18:03 707750590 1072270800 5361354 2 5361354 index:0,count:5361354,average:100,stdev:0|index:1,count:5361354,average:100,stdev:0 GSM1702741_r1 2.26 4.4 0.02 823051595 1061535096 791016817 1024034996 128.98 129.46 4739304 4129627 252.492 1649.214 160 21362 70.92 74.0 5073725 3360968 5073725 3360968 69.67 69.98 5073725 3302080 5073725 3178228 113495113 13.79 2.10 0 3.68 0 0.13 0 0.03 0 0.00 0 11.44 0 4739304 0 200 0 195.97 0 1.57 0 0.02 0 1.50 0 0.01 0 140.88 0 0.47 0 112584 0 5361354 0 197385 0 6908 0 1613 0 0 0 613529 0 764 0 0 0 12009 0 1536459 0 12617 0 1561849 0 84.72 0 4541919 0 57608 1503554 26.099743091237 5361354.0 4739304.0 112584.0 197385.0 6908.0 1613.0 0.0 613529.0 4541919.0 88.4 2.1 3.7 0.1 0.0 0.0 11.4 84.7 100 100 100.00 38 536135400 27.3 21.9 21.5 29.3 0.0 35.6 18.0 smartseq 250891 SRR2049502 SRP059035 SRS951869 SRX1047582 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702750: LC-MBT-15_SC42; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702750 GSM1702750 LC-MBT-15_SC42 1116467800 5582339 2015-06-05 16:18:03 736083981 1116467800 5582339 2 5582339 index:0,count:5582339,average:100,stdev:0|index:1,count:5582339,average:100,stdev:0 GSM1702750_r1 2.25 4.46 0.02 732295228 916688804 706014706 887189813 125.18 125.66 4578635 4116183 223.209 1356.326 111 22954 65.62 68.35 4907278 3004449 4907278 3004449 64.11 64.52 4907278 2935480 4907278 2835951 121837097 16.64 1.86 0 3.28 0 0.13 0 0.04 0 0.00 0 17.80 0 4578635 0 200 0 194.05 0 1.59 0 0.02 0 1.47 0 0.01 0 118.21 0 0.44 0 103926 0 5582339 0 183138 0 7460 0 2358 0 0 0 993886 0 536 0 0 0 10619 0 1292288 0 12721 0 1316164 0 78.74 0 4395497 0 53286 1192124 22.372180310025 5582339.0 4578635.0 103926.0 183138.0 7460.0 2358.0 0.0 993886.0 4395497.0 82.0 1.9 3.3 0.1 0.0 0.0 17.8 78.7 100 100 100.00 38 558233900 27.2 22.3 21.2 29.3 0.0 35.6 17.9 smartseq 250895 SRR2049503 SRP059035 SRS951868 SRX1047583 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702751: LC-MBT-15_SC45; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702751 GSM1702751 LC-MBT-15_SC45 1332817200 6664086 2015-06-05 16:18:03 877898749 1332817200 6664086 2 6664086 index:0,count:6664086,average:100,stdev:0|index:1,count:6664086,average:100,stdev:0 GSM1702751_r1 2.67 3.83 0.02 883337544 1143742602 851369428 1107131685 129.48 130.04 5503011 4891074 224.503 1270.219 111 27139 72.52 75.54 5901405 3990667 5901405 3990667 70.76 71.22 5901405 3894134 5901405 3762481 108988530 12.34 1.67 0 3.30 0 0.12 0 0.03 0 0.00 0 17.27 0 5503011 0 200 0 194.25 0 1.55 0 0.02 0 1.47 0 0.01 0 142.80 0 0.41 0 111094 0 6664086 0 219909 0 8041 0 2307 0 0 0 1150727 0 842 0 0 0 13033 0 1749763 0 13746 0 1777384 0 79.28 0 5283102 0 52595 1615034 30.706987356213 6664086.0 5503011.0 111094.0 219909.0 8041.0 2307.0 0.0 1150727.0 5283102.0 82.6 1.7 3.3 0.1 0.0 0.0 17.3 79.3 100 100 100.00 38 666408600 27.2 22.3 21.3 29.2 0.0 35.6 18.0 smartseq 251047 SRR2049529 SRP059035 SRS951842 SRX1047609 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702777: LC-MBT-15_SC85; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702777 GSM1702777 LC-MBT-15_SC85 1640284000 8201420 2015-06-05 16:18:03 1080282653 1640284000 8201420 2 8201420 index:0,count:8201420,average:100,stdev:0|index:1,count:8201420,average:100,stdev:0 GSM1702777_r1 3.43 4.24 0.03 1099118319 1405129016 1058769450 1358629304 127.84 128.32 6844316 6131289 223.454 1263.634 111 33954 67.34 70.13 7315520 4608738 7315520 4608738 65.89 65.99 7315520 4509947 7315520 4336483 181338256 16.50 1.58 0 3.33 0 0.10 0 0.05 0 0.00 0 16.40 0 6844316 0 200 0 194.41 0 1.57 0 0.02 0 1.48 0 0.01 0 191.72 0 0.40 0 129522 0 8201420 0 272874 0 8408 0 3739 0 0 0 1344957 0 803 0 0 0 14539 0 2014905 0 16142 0 2046389 0 80.13 0 6571442 0 49931 1869006 37.431775850674 8201420.0 6844316.0 129522.0 272874.0 8408.0 3739.0 0.0 1344957.0 6571442.0 83.5 1.6 3.3 0.1 0.0 0.0 16.4 80.1 100 100 100.00 38 820142000 27.3 22.4 21.1 29.2 0.0 35.8 18.6 smartseq 251075 SRR2049530 SRP059035 SRS951841 SRX1047610 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702778: LC-MBT-15_SC87; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702778 GSM1702778 LC-MBT-15_SC87 1195662600 5978313 2015-06-05 16:18:03 791129853 1195662600 5978313 2 5978313 index:0,count:5978313,average:100,stdev:0|index:1,count:5978313,average:100,stdev:0 GSM1702778_r1 2.72 4.01 0.02 889318542 1136654416 859470353 1102003918 127.81 128.22 5288343 4702368 239.512 1398.168 133 25014 67.65 70.23 5612998 3577817 5612998 3577817 66.15 66.45 5612998 3498106 5612998 3385146 137693249 15.48 1.97 0 3.25 0 0.15 0 0.04 0 0.00 0 11.35 0 5288343 0 200 0 195.79 0 1.56 0 0.02 0 1.46 0 0.01 0 154.83 0 0.44 0 117521 0 5978313 0 194228 0 8758 0 2610 0 0 0 678602 0 632 0 0 0 13200 0 1576900 0 13294 0 1604026 0 85.21 0 5094115 0 54023 1496261 27.696740277289 5978313.0 5288343.0 117521.0 194228.0 8758.0 2610.0 0.0 678602.0 5094115.0 88.5 2.0 3.2 0.1 0.0 0.0 11.4 85.2 100 100 100.00 38 597831300 27.6 21.6 21.1 29.7 0.0 35.6 18.0 smartseq 251079 SRR2049531 SRP059035 SRS951840 SRX1047611 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702779: LC-MBT-15_SC89; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702779 GSM1702779 LC-MBT-15_SC89 1758951200 8794756 2015-06-05 16:18:03 1163880447 1758951200 8794756 2 8794756 index:0,count:8794756,average:100,stdev:0|index:1,count:8794756,average:100,stdev:0 GSM1702779_r1 2.46 4.33 0.01 1408546115 1798395094 1362426293 1743894378 127.68 128.0 7988177 6967693 260.628 1283.484 164 34024 67.93 70.36 8442872 5426589 8442872 5426589 66.54 66.52 8442872 5315583 8442872 5130626 227347445 16.14 1.31 0 3.13 0 0.08 0 0.05 0 0.00 0 9.05 0 7988177 0 200 0 196.80 0 1.55 0 0.02 0 1.49 0 0.01 0 150.05 0 0.37 0 115083 0 8794756 0 275315 0 6729 0 4273 0 0 0 795577 0 1090 0 0 0 15233 0 2558530 0 20519 0 2595372 0 87.70 0 7712862 0 42865 2505916 58.460655546483 8794756.0 7988177.0 115083.0 275315.0 6729.0 4273.0 0.0 795577.0 7712862.0 90.8 1.3 3.1 0.1 0.0 0.0 9.0 87.7 100 100 100.00 38 879475600 27.7 21.8 21.5 29.1 0.0 35.8 18.7 smartseq 251095 SRR2049535 SRP059035 SRS951836 SRX1047615 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702783: LC-MBT-15_SC96; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702783 GSM1702783 LC-MBT-15_SC96 1752849400 8764247 2015-06-05 16:18:03 1152185152 1752849400 8764247 2 8764247 index:0,count:8764247,average:100,stdev:0|index:1,count:8764247,average:100,stdev:0 GSM1702783_r1 2.84 4.05 0.02 1348967516 1704932309 1306002859 1654513694 126.39 126.69 7928092 7024068 242.930 1239.244 152 36030 66.64 68.97 8367217 5283190 8367217 5283190 65.37 65.34 8367217 5182488 8367217 5004764 229090994 16.98 1.43 0 3.06 0 0.08 0 0.03 0 0.00 0 9.43 0 7928092 0 200 0 196.39 0 1.55 0 0.02 0 1.47 0 0.01 0 151.69 0 0.37 0 125232 0 8764247 0 268200 0 6603 0 2854 0 0 0 826698 0 1092 0 0 0 18642 0 2410162 0 19023 0 2448919 0 87.40 0 7659892 0 50638 2312279 45.662921126427 8764247.0 7928092.0 125232.0 268200.0 6603.0 2854.0 0.0 826698.0 7659892.0 90.5 1.4 3.1 0.1 0.0 0.0 9.4 87.4 100 100 100.00 38 876424700 27.8 21.7 21.3 29.3 0.0 35.9 18.8 smartseq 251099 SRR2049536 SRP059035 SRS951835 SRX1047616 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702784: LC-PT-45-mock; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702784 GSM1702784 LC-PT-45-mock 1886372200 9431861 2015-06-05 16:18:03 1282569774 1886372200 9431861 2 9431861 index:0,count:9431861,average:100,stdev:0|index:1,count:9431861,average:100,stdev:0 GSM1702784_r1 2.69 2.44 0.05 1387755637 1880248646 1297124324 1781007971 135.49 137.3 8253646 6911733 246.556 2244.996 144 35022 87.28 93.76 9675820 7203816 9675820 7203816 86.79 88.4 9675820 7162970 9675820 6791853 37700784 2.72 1.82 0 6.05 0 0.13 0 0.02 0 0.00 0 12.35 0 8253646 0 200 0 195.18 0 1.51 0 0.01 0 1.46 0 0.01 0 128.13 0 0.48 0 171206 0 9431861 0 570623 0 11913 0 1793 0 0 0 1164509 0 1580 0 0 0 24973 0 3804427 0 21819 0 3852799 0 81.46 0 7683023 0 131398 3626268 27.597589004399 9431861.0 8253646.0 171206.0 570623.0 11913.0 1793.0 0.0 1164509.0 7683023.0 87.5 1.8 6.0 0.1 0.0 0.0 12.3 81.5 100 100 100.00 38 943186100 26.0 23.4 23.1 27.6 0.0 35.4 18.9 smartseq 251106 SRR2049538 SRP059035 SRS951833 SRX1047618 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702786: LC-PT-45-Selumetinib_R3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702786 GSM1702786 LC-PT-45-Selumetinib_R3 1980582800 9902914 2015-06-05 16:18:03 1339746925 1980582800 9902914 2 9902914 index:0,count:9902914,average:100,stdev:0|index:1,count:9902914,average:100,stdev:0 GSM1702786_r1 3.0 2.71 0.05 1444208782 1943043296 1368436612 1859162654 134.54 135.86 8591279 7151659 247.737 2264.253 144 36213 87.31 92.49 9736933 7501267 9736933 7501267 86.26 87.55 9736933 7410518 9736933 7100136 49720184 3.44 1.91 0 4.86 0 0.14 0 0.02 0 0.00 0 13.09 0 8591279 0 200 0 195.33 0 1.51 0 0.01 0 1.49 0 0.01 0 117.27 0 0.45 0 188996 0 9902914 0 481167 0 13801 0 1814 0 0 0 1296020 0 1666 0 0 0 27099 0 3930684 0 21100 0 3980549 0 81.90 0 8110112 0 136691 3722563 27.233416976977 9902914.0 8591279.0 188996.0 481167.0 13801.0 1814.0 0.0 1296020.0 8110112.0 86.8 1.9 4.9 0.1 0.0 0.0 13.1 81.9 100 100 100.00 38 990291400 26.1 23.2 22.9 27.8 0.0 35.4 18.8 smartseq 251111 SRR2049539 SRP059035 SRS951832 SRX1047619 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702787: LC-PT-45-Selumetinib_R7; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702787 GSM1702787 LC-PT-45-Selumetinib_R7 2867547000 14337735 2015-06-05 16:18:03 1933402046 2867547000 14337735 2 14337735 index:0,count:14337735,average:100,stdev:0|index:1,count:14337735,average:100,stdev:0 GSM1702787_r1 2.47 2.89 0.05 2180845299 2941167745 2079292097 2827393463 134.86 135.98 12874068 10658147 250.066 1736.325 155 53209 88.99 93.6 14329304 11456036 14329304 11456036 87.71 88.76 14329304 11292370 14329304 10863476 63983023 2.93 1.42 0 4.43 0 0.11 0 0.02 0 0.00 0 10.07 0 12874068 0 200 0 195.80 0 1.53 0 0.01 0 1.48 0 0.01 0 177.99 0 0.38 0 203040 0 14337735 0 634802 0 16475 0 2767 0 0 0 1444425 0 2751 0 0 0 42658 0 6117615 0 32900 0 6195924 0 85.36 0 12239266 0 151884 5780492 38.058597350610 14337735.0 12874068.0 203040.0 634802.0 16475.0 2767.0 0.0 1444425.0 12239266.0 89.8 1.4 4.4 0.1 0.0 0.0 10.1 85.4 100 100 100.00 38 1433773500 26.4 23.1 22.8 27.7 0.0 35.6 19.3 smartseq 498124 SRR2049339 SRP059035 SRS951824 SRX1047419 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702587: H358_Pooled; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702587 GSM1702587 H358_Pooled 1994597000 9972985 2015-06-05 16:18:03 1425499330 1994597000 9972985 2 9972985 index:0,count:9972985,average:100,stdev:0|index:1,count:9972985,average:100,stdev:0 GSM1702587_r1 2.14 2.97 0.07 1426094287 1901346113 1310731015 1794629163 133.33 136.92 8499210 7045514 249.300 1693.468 139 32969 85.96 93.92 10836393 7306224 10836393 7306224 85.76 88.55 10836393 7289160 10836393 6888217 34112518 2.39 1.69 0 7.22 0 0.27 0 0.05 0 0.00 0 14.46 0 8499210 0 200 0 194.77 0 1.48 0 0.01 0 1.44 0 0.01 0 128.68 0 0.63 0 168872 0 9972985 0 720334 0 26791 0 5339 0 0 0 1441645 0 2127 0 0 0 29458 0 3987635 0 24526 0 4043746 0 78.00 0 7778876 0 129264 3789855 29.318719829187 9972985.0 8499210.0 168872.0 720334.0 26791.0 5339.0 0.0 1441645.0 7778876.0 85.2 1.7 7.2 0.3 0.1 0.0 14.5 78.0 100 100 100.00 38 997298500 25.9 23.7 23.1 27.3 0.0 34.8 18.4 smartseq 498181 SRR2049340 SRP059035 SRS951823 SRX1047420 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702588: H358_SC05; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702588 GSM1702588 H358_SC05 1127275400 5636377 2015-06-05 16:18:03 737566424 1127275400 5636377 2 5636377 index:0,count:5636377,average:100,stdev:0|index:1,count:5636377,average:100,stdev:0 GSM1702588_r1 2.71 3.24 0.16 740470080 936487932 712814547 904838307 126.47 126.94 4470029 3943252 244.723 1306.676 100 18591 62.98 65.63 4794035 2815149 4794035 2815149 61.71 61.72 4794035 2758342 4794035 2647479 148474800 20.05 1.43 0 3.21 0 0.16 0 0.07 0 0.00 0 20.46 0 4470029 0 200 0 194.83 0 1.53 0 0.02 0 1.47 0 0.01 0 142.89 0 0.35 0 80488 0 5636377 0 180770 0 9270 0 3841 0 0 0 1153237 0 982 0 0 0 10681 0 1328510 0 10261 0 1350434 0 76.10 0 4289259 0 43188 1260620 29.189126609243 5636377.0 4470029.0 80488.0 180770.0 9270.0 3841.0 0.0 1153237.0 4289259.0 79.3 1.4 3.2 0.2 0.1 0.0 20.5 76.1 100 100 100.00 38 563637700 27.2 22.8 21.2 28.7 0.0 35.6 18.1 smartseq 498188 SRR2049341 SRP059035 SRS951822 SRX1047421 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702589: H358_SC09; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702589 GSM1702589 H358_SC09 867865800 4339329 2015-06-05 16:18:03 575565601 867865800 4339329 2 4339329 index:0,count:4339329,average:100,stdev:0|index:1,count:4339329,average:100,stdev:0 GSM1702589_r1 2.95 3.41 0.1 742630820 940630705 718554290 913798692 126.66 127.17 4004427 3340426 312.245 1720.691 183 12203 67.68 70.06 4252508 2710320 4252508 2710320 66.35 66.49 4252508 2656872 4252508 2572261 125438817 16.89 1.51 0 3.13 0 0.12 0 0.07 0 0.00 0 7.52 0 4004427 0 200 0 197.32 0 1.56 0 0.02 0 1.48 0 0.01 0 132.39 0 0.37 0 65519 0 4339329 0 135884 0 5240 0 3141 0 0 0 326521 0 886 0 0 0 10383 0 1340266 0 10070 0 1361605 0 89.15 0 3868543 0 49483 1356478 27.413010528869 4339329.0 4004427.0 65519.0 135884.0 5240.0 3141.0 0.0 326521.0 3868543.0 92.3 1.5 3.1 0.1 0.1 0.0 7.5 89.2 100 100 100.00 38 433932900 27.5 22.1 21.8 28.6 0.0 35.8 18.9 smartseq 498197 SRR2049342 SRP059035 SRS951821 SRX1047422 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702590: H358_SC10; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702590 GSM1702590 H358_SC10 878075200 4390376 2015-06-05 16:18:03 576176110 878075200 4390376 2 4390376 index:0,count:4390376,average:100,stdev:0|index:1,count:4390376,average:100,stdev:0 GSM1702590_r1 2.82 3.63 0.15 752173562 938655995 728104915 912086741 124.79 125.27 4044825 3428529 314.143 1640.268 198 12355 63.74 65.94 4293196 2578071 4293196 2578071 62.76 62.82 4293196 2538691 4293196 2456036 149034999 19.81 1.53 0 3.08 0 0.15 0 0.06 0 0.00 0 7.66 0 4044825 0 200 0 197.39 0 1.54 0 0.02 0 1.51 0 0.01 0 192.75 0 0.36 0 67247 0 4390376 0 135188 0 6598 0 2792 0 0 0 336161 0 761 0 0 0 9088 0 1225306 0 9281 0 1244436 0 89.05 0 3909637 0 44472 1249528 28.096959884871 4390376.0 4044825.0 67247.0 135188.0 6598.0 2792.0 0.0 336161.0 3909637.0 92.1 1.5 3.1 0.2 0.1 0.0 7.7 89.1 100 100 100.00 38 439037600 27.8 21.7 21.4 29.1 0.0 35.9 18.7 smartseq 498204 SRR2049343 SRP059035 SRS951820 SRX1047423 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702591: H358_SC11; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702591 GSM1702591 H358_SC11 847991200 4239956 2015-06-05 16:18:03 557222901 847991200 4239956 2 4239956 index:0,count:4239956,average:100,stdev:0|index:1,count:4239956,average:100,stdev:0 GSM1702591_r1 3.39 2.87 0.08 722397600 913190180 696856677 884340284 126.41 126.9 3903563 3350115 306.402 1449.328 195 12491 62.94 65.34 4154154 2456739 4154154 2456739 62.2 62.21 4154154 2427881 4154154 2339086 141092576 19.53 1.45 0 3.39 0 0.16 0 0.08 0 0.00 0 7.69 0 3903563 0 200 0 197.34 0 1.55 0 0.02 0 1.50 0 0.01 0 151.13 0 0.36 0 61499 0 4239956 0 143689 0 6926 0 3575 0 0 0 325892 0 762 0 0 0 8660 0 1120123 0 9794 0 1139339 0 88.68 0 3759874 0 41569 1141848 27.468738723568 4239956.0 3903563.0 61499.0 143689.0 6926.0 3575.0 0.0 325892.0 3759874.0 92.1 1.5 3.4 0.2 0.1 0.0 7.7 88.7 100 100 100.00 38 423995600 27.7 21.8 21.6 28.9 0.0 35.9 18.8 smartseq 498213 SRR2049344 SRP059035 SRS951819 SRX1047424 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702592: H358_SC12; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702592 GSM1702592 H358_SC12 1122335200 5611676 2015-06-05 16:18:03 733123981 1122335200 5611676 2 5611676 index:0,count:5611676,average:100,stdev:0|index:1,count:5611676,average:100,stdev:0 GSM1702592_r1 3.1 3.59 0.1 892043444 1125469695 858742140 1089132499 126.17 126.83 5081778 4456031 262.985 1318.151 165 20775 64.77 67.42 5443473 3291455 5443473 3291455 63.68 63.8 5443473 3236146 5443473 3114640 157092338 17.61 1.45 0 3.56 0 0.14 0 0.10 0 0.00 0 9.20 0 5081778 0 200 0 196.78 0 1.52 0 0.02 0 1.46 0 0.01 0 204.06 0 0.34 0 81465 0 5611676 0 199971 0 7909 0 5702 0 0 0 516287 0 951 0 0 0 12541 0 1598209 0 12222 0 1623923 0 86.99 0 4881807 0 48165 1575822 32.717159763314 5611676.0 5081778.0 81465.0 199971.0 7909.0 5702.0 0.0 516287.0 4881807.0 90.6 1.5 3.6 0.1 0.1 0.0 9.2 87.0 100 100 100.00 38 561167600 27.5 22.1 21.7 28.7 0.0 35.9 19.0 smartseq 498220 SRR2049345 SRP059035 SRS951818 SRX1047425 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702593: H358_SC14; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702593 GSM1702593 H358_SC14 1125123800 5625619 2015-06-05 16:18:03 736805149 1125123800 5625619 2 5625619 index:0,count:5625619,average:100,stdev:0|index:1,count:5625619,average:100,stdev:0 GSM1702593_r1 3.16 3.28 0.12 749475864 952464403 720740281 920087431 127.08 127.66 4512524 3925265 246.244 1340.390 112 17995 67.39 70.3 4843647 3040934 4843647 3040934 66.31 66.45 4843647 2992300 4843647 2874355 123834259 16.52 1.39 0 3.32 0 0.15 0 0.07 0 0.00 0 19.57 0 4512524 0 200 0 194.88 0 1.53 0 0.01 0 1.48 0 0.01 0 180.82 0 0.34 0 78334 0 5625619 0 186812 0 8286 0 4002 0 0 0 1100807 0 982 0 0 0 11753 0 1517123 0 10419 0 1540277 0 76.89 0 4325712 0 45641 1437500 31.495804211126 5625619.0 4512524.0 78334.0 186812.0 8286.0 4002.0 0.0 1100807.0 4325712.0 80.2 1.4 3.3 0.1 0.1 0.0 19.6 76.9 100 100 100.00 38 562561900 27.2 23.0 21.4 28.5 0.0 35.7 18.5 smartseq 498228 SRR2049346 SRP059035 SRS951817 SRX1047426 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702594: H358_SC16; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702594 GSM1702594 H358_SC16 1049371400 5246857 2015-06-05 16:18:03 688867791 1049371400 5246857 2 5246857 index:0,count:5246857,average:100,stdev:0|index:1,count:5246857,average:100,stdev:0 GSM1702594_r1 3.31 3.3 0.12 722326284 908898517 691915177 874845764 125.83 126.44 4291423 3762966 257.273 1283.184 120 16037 64.31 67.38 4633440 2760022 4633440 2760022 63.46 63.57 4633440 2723193 4633440 2604066 126613453 17.53 1.48 0 3.72 0 0.16 0 0.06 0 0.00 0 18.00 0 4291423 0 200 0 195.17 0 1.52 0 0.02 0 1.46 0 0.01 0 165.69 0 0.34 0 77574 0 5246857 0 195260 0 8269 0 2900 0 0 0 944265 0 768 0 0 0 9216 0 1295942 0 10048 0 1315974 0 78.07 0 4096163 0 40889 1240796 30.345471887305 5246857.0 4291423.0 77574.0 195260.0 8269.0 2900.0 0.0 944265.0 4096163.0 81.8 1.5 3.7 0.2 0.1 0.0 18.0 78.1 100 100 100.00 38 524685700 27.6 22.3 21.1 29.0 0.0 35.6 18.2 smartseq 498236 SRR2049347 SRP059035 SRS951816 SRX1047427 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702595: H358_SC17; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702595 GSM1702595 H358_SC17 987062000 4935310 2015-06-05 16:18:03 656386455 987062000 4935310 2 4935310 index:0,count:4935310,average:100,stdev:0|index:1,count:4935310,average:100,stdev:0 GSM1702595_r1 3.73 3.45 0.08 681283951 847655959 657300101 821474087 124.42 124.98 4039614 3581094 253.986 1279.136 111 15169 60.9 63.3 4327606 2460089 4327606 2460089 59.92 60.06 4327606 2420483 4327606 2334175 137556026 20.19 1.45 0 3.10 0 0.14 0 0.08 0 0.00 0 17.94 0 4039614 0 200 0 195.28 0 1.54 0 0.02 0 1.50 0 0.01 0 143.28 0 0.39 0 71756 0 4935310 0 153151 0 6795 0 3742 0 0 0 885159 0 716 0 0 0 8558 0 1114731 0 9351 0 1133356 0 78.75 0 3886463 0 42010 1064973 25.350464175196 4935310.0 4039614.0 71756.0 153151.0 6795.0 3742.0 0.0 885159.0 3886463.0 81.9 1.5 3.1 0.1 0.1 0.0 17.9 78.7 100 100 100.00 38 493531000 27.8 22.2 21.1 29.0 0.0 35.7 18.3 smartseq 498252 SRR2049349 SRP059035 SRS951814 SRX1047429 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702597: H358_SC23; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell line;;H358|source_name;;human lung cancer cell line GEO Accession;;GSM1702597 GSM1702597 H358_SC23 1119547200 5597736 2015-06-05 16:18:03 731391909 1119547200 5597736 2 5597736 index:0,count:5597736,average:100,stdev:0|index:1,count:5597736,average:100,stdev:0 GSM1702597_r1 4.88 3.41 0.14 893488453 1139484892 860245946 1102777597 127.53 128.19 5075428 4436619 266.181 1343.523 175 20458 66.72 69.46 5438081 3386474 5438081 3386474 65.66 65.95 5438081 3332570 5438081 3215774 145678790 16.30 1.46 0 3.57 0 0.18 0 0.09 0 0.00 0 9.06 0 5075428 0 200 0 196.82 0 1.52 0 0.02 0 1.46 0 0.01 0 169.34 0 0.34 0 81538 0 5597736 0 199658 0 10249 0 5099 0 0 0 506960 0 919 0 0 0 11459 0 1579177 0 11972 0 1603527 0 87.10 0 4875770 0 50542 1547474 30.617585374540 5597736.0 5075428.0 81538.0 199658.0 10249.0 5099.0 0.0 506960.0 4875770.0 90.7 1.5 3.6 0.2 0.1 0.0 9.1 87.1 100 100 100.00 38 559773600 27.7 21.9 21.5 28.9 0.0 35.9 19.0 smartseq 499868 SRR2049413 SRP059035 SRS951960 SRX1047493 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702661: LC-PT-45_SC50; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702661 GSM1702661 LC-PT-45_SC50 2695425178 13343689 2015-06-05 16:18:03 1824977216 2695425178 13343689 2 13343689 index:0,count:13343689,average:101,stdev:0|index:1,count:13343689,average:101,stdev:0 GSM1702661_r1 3.49 3.18 0.09 2067604415 2699096326 1989656559 2616187797 130.54 131.49 11735967 10710001 242.784 1062.238 160 57865 67.31 70.1 12721724 7899773 12721724 7899773 66.46 66.82 12721724 7799434 12721724 7530473 339314769 16.41 1.59 0 3.50 0 0.26 0 0.07 0 0.00 0 11.72 0 11735967 0 202 0 198.77 0 1.48 0 0.02 0 1.53 0 0.01 0 164.51 0 0.44 0 211509 0 13343689 0 466995 0 34186 0 9025 0 0 0 1564511 0 1127 0 0 0 19395 0 3134400 0 23071 0 3177993 0 84.45 0 11268972 0 47701 3061589 64.182910211526 13343689.0 11735967.0 211509.0 466995.0 34186.0 9025.0 0.0 1564511.0 11268972.0 88.0 1.6 3.5 0.3 0.1 0.0 11.7 84.5 101 101 101.00 38 1347712589 28.5 21.0 20.8 29.7 0.0 35.0 17.1 smartseq 499876 SRR2049414 SRP059035 SRS951957 SRX1047494 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702662: LC-PT-45_SC53; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702662 GSM1702662 LC-PT-45_SC53 2839317252 14056026 2015-06-05 16:18:03 1922000766 2839317252 14056026 2 14056026 index:0,count:14056026,average:101,stdev:0|index:1,count:14056026,average:101,stdev:0 GSM1702662_r1 8.61 3.5 0.08 2161509059 2966928788 2078644565 2884266389 137.26 138.76 12300391 11163713 239.059 1085.359 163 61396 77.27 80.53 13485138 9504804 13485138 9504804 75.86 76.79 13485138 9331517 13485138 9063553 225699116 10.44 1.52 0 3.54 0 0.29 0 0.06 0 0.00 0 12.15 0 12300391 0 202 0 198.69 0 1.49 0 0.02 0 1.50 0 0.01 0 142.54 0 0.43 0 213647 0 14056026 0 497477 0 40070 0 7935 0 0 0 1707630 0 3076 0 0 0 21996 0 3452781 0 24201 0 3502054 0 83.97 0 11802914 0 41221 3383649 82.085563183814 14056026.0 12300391.0 213647.0 497477.0 40070.0 7935.0 0.0 1707630.0 11802914.0 87.5 1.5 3.5 0.3 0.1 0.0 12.1 84.0 101 101 101.00 38 1419658626 28.3 21.2 21.0 29.5 0.0 34.9 16.9 smartseq 499884 SRR2049415 SRP059035 SRS951956 SRX1047495 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702663: LC-PT-45_SC54; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702663 GSM1702663 LC-PT-45_SC54 2083681914 10315257 2015-06-05 16:18:03 1413691104 2083681914 10315257 2 10315257 index:0,count:10315257,average:101,stdev:0|index:1,count:10315257,average:101,stdev:0 GSM1702663_r1 9.34 3.07 0.09 1581339592 2202971994 1516501894 2131758945 139.31 140.57 8972094 8065506 243.337 1026.543 176 44671 77.47 80.97 9783416 6950264 9783416 6950264 76.64 77.18 9783416 6876192 9783416 6625290 157761750 9.98 1.62 0 3.76 0 0.21 0 0.04 0 0.00 0 12.77 0 8972094 0 202 0 198.55 0 1.44 0 0.02 0 1.43 0 0.01 0 129.84 0 0.45 0 167353 0 10315257 0 387842 0 22147 0 3702 0 0 0 1317314 0 972 0 0 0 16626 0 2765215 0 18581 0 2801394 0 83.22 0 8584252 0 34363 2746099 79.914413758985 10315257.0 8972094.0 167353.0 387842.0 22147.0 3702.0 0.0 1317314.0 8584252.0 87.0 1.6 3.8 0.2 0.0 0.0 12.8 83.2 101 101 101.00 38 1041840957 28.1 21.4 21.2 29.3 0.0 34.9 16.8 smartseq 499892 SRR2049416 SRP059035 SRS951955 SRX1047496 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702664: LC-PT-45_SC58; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702664 GSM1702664 LC-PT-45_SC58 1136691370 5627185 2015-06-05 16:18:03 763010284 1136691370 5627185 2 5627185 index:0,count:5627185,average:101,stdev:0|index:1,count:5627185,average:101,stdev:0 GSM1702664_r1 8.56 2.46 0.08 838359964 1134569346 810289323 1106194887 135.33 136.52 4836342 4476395 230.968 941.836 165 26986 68.87 71.44 5212340 3330822 5212340 3330822 67.49 68.1 5212340 3264087 5212340 3174815 137111300 16.35 1.75 0 3.09 0 0.31 0 0.06 0 0.00 0 13.68 0 4836342 0 202 0 198.44 0 1.48 0 0.02 0 1.47 0 0.01 0 133.28 0 0.45 0 98734 0 5627185 0 174095 0 17594 0 3555 0 0 0 769694 0 534 0 0 0 6962 0 1116603 0 8570 0 1132669 0 82.85 0 4662247 0 37346 1097611 29.390322926150 5627185.0 4836342.0 98734.0 174095.0 17594.0 3555.0 0.0 769694.0 4662247.0 85.9 1.8 3.1 0.3 0.1 0.0 13.7 82.9 101 101 101.00 38 568345685 28.6 21.0 20.5 29.9 0.0 35.0 16.8 smartseq 499972 SRR2049420 SRP059035 SRS951951 SRX1047500 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702668: LC-PT-45_SC72; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702668 GSM1702668 LC-PT-45_SC72 2737906788 13553994 2015-06-05 16:18:03 1857046560 2737906788 13553994 2 13553994 index:0,count:13553994,average:101,stdev:0|index:1,count:13553994,average:101,stdev:0 GSM1702668_r1 4.5 3.21 0.06 2167132487 2903132899 2073008670 2795331003 133.96 134.84 12288268 10884501 242.674 1088.149 175 61087 74.02 77.54 13294864 9096140 13294864 9096140 72.66 73.14 13294864 8928565 13294864 8579872 281892899 13.01 1.22 0 4.11 0 0.17 0 0.03 0 0.00 0 9.14 0 12288268 0 202 0 198.91 0 1.49 0 0.01 0 1.50 0 0.01 0 186.24 0 0.42 0 165412 0 13553994 0 557059 0 22466 0 4506 0 0 0 1238754 0 2478 0 0 0 29688 0 4499938 0 30448 0 4562552 0 86.55 0 11731209 0 46229 4374532 94.627441649181 13553994.0 12288268.0 165412.0 557059.0 22466.0 4506.0 0.0 1238754.0 11731209.0 90.7 1.2 4.1 0.2 0.0 0.0 9.1 86.6 101 101 101.00 38 1368953394 27.8 21.8 21.7 28.7 0.0 35.5 18.3 smartseq 499980 SRR2049421 SRP059035 SRS951950 SRX1047501 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702669: LC-PT-45_SC76; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702669 GSM1702669 LC-PT-45_SC76 2434198980 12050490 2015-06-05 16:18:03 1642506369 2434198980 12050490 2 12050490 index:0,count:12050490,average:101,stdev:0|index:1,count:12050490,average:101,stdev:0 GSM1702669_r1 5.77 2.37 0.1 1887815904 2480114876 1823013630 2410001817 131.37 132.2 10767949 9939958 237.183 898.362 163 54485 64.58 66.99 11578737 6953690 11578737 6953690 63.59 63.83 11578737 6847186 11578737 6625129 359505988 19.04 1.42 0 3.22 0 0.17 0 0.06 0 0.00 0 10.41 0 10767949 0 202 0 198.88 0 1.48 0 0.02 0 1.49 0 0.01 0 177.79 0 0.41 0 171650 0 12050490 0 387952 0 20207 0 7638 0 0 0 1254696 0 1304 0 0 0 18215 0 2661176 0 20681 0 2701376 0 86.14 0 10379997 0 35908 2598729 72.371866993428 12050490.0 10767949.0 171650.0 387952.0 20207.0 7638.0 0.0 1254696.0 10379997.0 89.4 1.4 3.2 0.2 0.1 0.0 10.4 86.1 101 101 101.00 38 1217099490 28.5 21.1 20.9 29.5 0.0 35.5 17.9 smartseq 499988 SRR2049422 SRP059035 SRS951949 SRX1047502 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702670: LC-PT-45_SC77; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702670 GSM1702670 LC-PT-45_SC77 1941001436 9608918 2015-06-05 16:18:03 1301907705 1941001436 9608918 2 9608918 index:0,count:9608918,average:101,stdev:0|index:1,count:9608918,average:101,stdev:0 GSM1702670_r1 4.51 3.12 0.13 1502221438 1983831736 1452922290 1932044075 132.06 132.98 8561541 7705315 238.074 1129.194 160 43314 73.51 76.15 9149305 6293531 9149305 6293531 72.3 72.8 9149305 6189905 9149305 6016518 197796284 13.17 1.51 0 3.09 0 0.24 0 0.12 0 0.00 0 10.55 0 8561541 0 202 0 198.77 0 1.51 0 0.01 0 1.49 0 0.01 0 136.73 0 0.42 0 145036 0 9608918 0 297335 0 22620 0 11130 0 0 0 1013627 0 1416 0 0 0 15839 0 2606175 0 19193 0 2642623 0 86.01 0 8264206 0 46911 2541970 54.187077657692 9608918.0 8561541.0 145036.0 297335.0 22620.0 11130.0 0.0 1013627.0 8264206.0 89.1 1.5 3.1 0.2 0.1 0.0 10.5 86.0 101 101 101.00 38 970500718 28.5 21.0 20.9 29.6 0.0 35.7 18.1 smartseq 499997 SRR2049423 SRP059035 SRS951948 SRX1047503 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702671: LC-PT-45_SC79; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702671 GSM1702671 LC-PT-45_SC79 2298654758 11379479 2015-06-05 16:18:03 1548329446 2298654758 11379479 2 11379479 index:0,count:11379479,average:101,stdev:0|index:1,count:11379479,average:101,stdev:0 GSM1702671_r1 10.42 2.53 0.21 1731464792 2168336669 1668290452 2102597658 125.23 126.03 9906291 9497960 232.780 679.950 163 51844 46.07 47.93 10708562 4564257 10708562 4564257 45.61 45.55 10708562 4517821 10708562 4337818 529261496 30.57 1.90 0 3.37 0 0.25 0 0.08 0 0.00 0 12.62 0 9906291 0 202 0 198.71 0 1.56 0 0.02 0 1.48 0 0.01 0 151.17 0 0.43 0 216373 0 11379479 0 383135 0 28460 0 8633 0 0 0 1436095 0 888 0 0 0 11352 0 1375004 0 22724 0 1409968 0 83.69 0 9523156 0 22453 1421036 63.289359996437 11379479.0 9906291.0 216373.0 383135.0 28460.0 8633.0 0.0 1436095.0 9523156.0 87.1 1.9 3.4 0.3 0.1 0.0 12.6 83.7 101 101 101.00 38 1149327379 28.2 21.2 21.0 29.5 0.0 35.4 17.4 smartseq 500004 SRR2049424 SRP059035 SRS951947 SRX1047504 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702672: LC-PT-45_SC87; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702672 GSM1702672 LC-PT-45_SC87 933423820 4620910 2015-06-05 16:18:03 633616166 933423820 4620910 2 4620910 index:0,count:4620910,average:101,stdev:0|index:1,count:4620910,average:101,stdev:0 GSM1702672_r1 6.31 2.28 0.07 770565352 1051683331 742214508 1020180150 136.48 137.45 4367299 3857205 261.611 1392.637 159 22036 74.5 77.58 4701179 3253725 4701179 3253725 73.04 73.65 4701179 3189741 4701179 3088963 98500294 12.78 1.99 0 3.75 0 0.19 0 0.03 0 0.00 0 5.27 0 4367299 0 202 0 198.45 0 1.47 0 0.01 0 1.56 0 0.01 0 147.21 0 0.43 0 91778 0 4620910 0 173054 0 8835 0 1481 0 0 0 243295 0 642 0 0 0 8942 0 1310562 0 10882 0 1331028 0 90.77 0 4194245 0 44602 1296172 29.060849289270 4620910.0 4367299.0 91778.0 173054.0 8835.0 1481.0 0.0 243295.0 4194245.0 94.5 2.0 3.7 0.2 0.0 0.0 5.3 90.8 101 101 101.00 38 466711910 28.0 21.9 21.6 28.4 0.0 36.4 22.8 smartseq 500013 SRR2049425 SRP059035 SRS951946 SRX1047505 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702673: LC-PT-45_SC88; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702673 GSM1702673 LC-PT-45_SC88 1243622898 6156549 2015-06-05 16:18:03 842675009 1243622898 6156549 2 6156549 index:0,count:6156549,average:101,stdev:0|index:1,count:6156549,average:101,stdev:0 GSM1702673_r1 15.32 2.53 0.15 1000583300 1337595622 965238489 1301072614 133.68 134.79 5742478 5502284 247.769 724.489 150 26709 53.29 55.4 6288392 3060144 6288392 3060144 52.93 53.19 6288392 3039340 6288392 2938124 265008990 26.49 2.56 0 3.56 0 0.25 0 0.10 0 0.00 0 6.38 0 5742478 0 202 0 198.44 0 1.48 0 0.02 0 1.46 0 0.01 0 116.04 0 0.39 0 157843 0 6156549 0 219166 0 15125 0 5955 0 0 0 392991 0 339 0 0 0 6463 0 747468 0 12501 0 766771 0 89.71 0 5523312 0 21277 751631 35.325985806270 6156549.0 5742478.0 157843.0 219166.0 15125.0 5955.0 0.0 392991.0 5523312.0 93.3 2.6 3.6 0.2 0.1 0.0 6.4 89.7 101 101 101.00 38 621811449 28.2 21.6 21.4 28.8 0.0 36.5 22.9 smartseq 500020 SRR2049426 SRP059035 SRS951945 SRX1047506 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702674: LC-PT-45_SC91; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702674 GSM1702674 LC-PT-45_SC91 1443041742 7143771 2015-06-05 16:18:03 976963013 1443041742 7143771 2 7143771 index:0,count:7143771,average:101,stdev:0|index:1,count:7143771,average:101,stdev:0 GSM1702674_r1 5.23 3.24 0.11 1200386917 1551995077 1159065991 1509351072 129.29 130.22 6775094 6161916 261.368 1065.132 152 28274 62.95 65.32 7312078 4264822 7312078 4264822 62.07 62.43 7312078 4205520 7312078 4076260 247021803 20.58 1.74 0 3.45 0 0.17 0 0.06 0 0.00 0 4.92 0 6775094 0 202 0 198.97 0 1.53 0 0.02 0 1.56 0 0.01 0 212.54 0 0.36 0 124298 0 7143771 0 246105 0 12456 0 4529 0 0 0 351692 0 1017 0 0 0 10699 0 1656261 0 13852 0 1681829 0 91.39 0 6528989 0 35970 1628812 45.282513205449 7143771.0 6775094.0 124298.0 246105.0 12456.0 4529.0 0.0 351692.0 6528989.0 94.8 1.7 3.4 0.2 0.1 0.0 4.9 91.4 101 101 101.00 38 721520871 28.7 21.3 21.0 29.0 0.0 36.5 23.1 smartseq 500028 SRR2049427 SRP059035 SRS951944 SRX1047507 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702675: LC-PT-45_SC93; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702675 GSM1702675 LC-PT-45_SC93 1492954124 7390862 2015-06-05 16:18:03 1014318673 1492954124 7390862 2 7390862 index:0,count:7390862,average:101,stdev:0|index:1,count:7390862,average:101,stdev:0 GSM1702675_r1 8.75 4.09 0.13 1224696944 1568883880 1183678798 1526806186 128.1 128.99 6971377 6532794 252.394 855.276 155 30659 54.18 56.18 7584928 3777196 7584928 3777196 53.58 53.71 7584928 3735192 7584928 3611427 329559240 26.91 1.85 0 3.35 0 0.13 0 0.09 0 0.00 0 5.45 0 6971377 0 202 0 198.95 0 1.53 0 0.02 0 1.46 0 0.01 0 211.17 0 0.36 0 136852 0 7390862 0 247422 0 9809 0 6974 0 0 0 402702 0 537 0 0 0 10176 0 1280806 0 13131 0 1304650 0 90.98 0 6723955 0 27094 1257633 46.417398686056 7390862.0 6971377.0 136852.0 247422.0 9809.0 6974.0 0.0 402702.0 6723955.0 94.3 1.9 3.3 0.1 0.1 0.0 5.4 91.0 101 101 101.00 38 746477062 28.5 21.5 21.2 28.8 0.0 36.5 23.1 smartseq 500036 SRR2049428 SRP059035 SRS951943 SRX1047508 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702676: LC-PT-45_SC94; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702676 GSM1702676 LC-PT-45_SC94 2549555524 12621562 2015-06-05 16:18:03 1710162264 2549555524 12621562 2 12621562 index:0,count:12621562,average:101,stdev:0|index:1,count:12621562,average:101,stdev:0 GSM1702676_r1 7.61 2.46 0.07 1878375904 2487462268 1801222978 2406699393 132.43 133.61 10850004 9980010 231.728 1109.641 165 64285 64.82 67.83 11906259 7033279 11906259 7033279 63.63 64.22 11906259 6903865 11906259 6658797 349381105 18.60 1.96 0 3.81 0 0.34 0 0.05 0 0.00 0 13.65 0 10850004 0 202 0 198.18 0 1.50 0 0.01 0 1.53 0 0.01 0 113.31 0 0.48 0 246765 0 12621562 0 481157 0 43302 0 5719 0 0 0 1722537 0 1280 0 0 0 20126 0 2757314 0 26850 0 2805570 0 82.15 0 10368847 0 63195 2709921 42.881889389983 12621562.0 10850004.0 246765.0 481157.0 43302.0 5719.0 0.0 1722537.0 10368847.0 86.0 2.0 3.8 0.3 0.0 0.0 13.6 82.2 101 101 101.00 38 1274777762 27.8 21.5 21.3 29.3 0.0 35.2 17.2 smartseq 500044 SRR2049429 SRP059035 SRS951942 SRX1047509 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702677: LC-PT-45_SC95; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702677 GSM1702677 LC-PT-45_SC95 3089645752 15295276 2015-06-05 16:18:03 2083904657 3089645752 15295276 2 15295276 index:0,count:15295276,average:101,stdev:0|index:1,count:15295276,average:101,stdev:0 GSM1702677_r1 10.78 2.55 0.16 2290434731 3061742075 2196576076 2959933988 133.68 134.75 13177792 12294968 232.424 916.097 160 69188 62.42 65.3 14468840 8226125 14468840 8226125 61.89 62.29 14468840 8155467 14468840 7847101 457277337 19.96 2.02 0 3.79 0 0.32 0 0.07 0 0.00 0 13.46 0 13177792 0 202 0 198.36 0 1.49 0 0.02 0 1.49 0 0.01 0 121.55 0 0.46 0 308924 0 15295276 0 580089 0 48665 0 9993 0 0 0 2058826 0 1065 0 0 0 22269 0 2947016 0 29180 0 2999530 0 82.36 0 12597703 0 62808 2898971 46.156078843459 15295276.0 13177792.0 308924.0 580089.0 48665.0 9993.0 0.0 2058826.0 12597703.0 86.2 2.0 3.8 0.3 0.1 0.0 13.5 82.4 101 101 101.00 38 1544822876 28.0 21.3 21.2 29.4 0.0 35.2 17.1 smartseq 500100 SRR2049430 SRP059035 SRS951941 SRX1047510 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702678: LC-PT-45-Re_BEZ235; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702678 GSM1702678 LC-PT-45-Re_BEZ235 2475539600 12377698 2015-06-05 16:18:03 1635088947 2475539600 12377698 2 12377698 index:0,count:12377698,average:100,stdev:0|index:1,count:12377698,average:100,stdev:0 GSM1702678_r1 3.58 2.8 0.05 2046549957 2802648500 1947070967 2691126486 136.95 138.21 11304155 8670140 324.744 2302.575 164 30027 86.11 90.72 12598017 9734130 12598017 9734130 85.54 86.36 12598017 9669886 12598017 9266637 89502482 4.37 1.52 0 4.64 0 0.11 0 0.03 0 0.00 0 8.53 0 11304155 0 200 0 196.62 0 1.56 0 0.01 0 1.52 0 0.01 0 183.37 0 0.39 0 188636 0 12377698 0 573775 0 14074 0 3819 0 0 0 1055650 0 1956 0 0 0 36859 0 4903179 0 27187 0 4969181 0 86.69 0 10730380 0 147496 4883180 33.107202907197 12377698.0 11304155.0 188636.0 573775.0 14074.0 3819.0 0.0 1055650.0 10730380.0 91.3 1.5 4.6 0.1 0.0 0.0 8.5 86.7 100 100 100.00 38 1237769800 26.5 23.1 22.9 27.5 0.0 35.9 19.5 smartseq 500108 SRR2049431 SRP059035 SRS951940 SRX1047511 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702679: LC-PT-45-Re_BKM120; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702679 GSM1702679 LC-PT-45-Re_BKM120 1162012000 5810060 2015-06-05 16:18:03 754545228 1162012000 5810060 2 5810060 index:0,count:5810060,average:100,stdev:0|index:1,count:5810060,average:100,stdev:0 GSM1702679_r1 4.1 2.72 0.05 894033334 1223488613 848377790 1173892497 136.85 138.37 5170529 4278835 268.583 1975.572 159 19777 84.6 89.43 5847487 4374115 5847487 4374115 83.7 84.84 5847487 4327811 5847487 4149656 43540560 4.87 1.95 0 4.81 0 0.13 0 0.03 0 0.00 0 10.85 0 5170529 0 200 0 195.93 0 1.54 0 0.01 0 1.51 0 0.01 0 164.69 0 0.42 0 113444 0 5810060 0 279342 0 7606 0 1712 0 0 0 630213 0 926 0 0 0 15879 0 2173279 0 13013 0 2203097 0 84.18 0 4891187 0 117042 2104581 17.981416927257 5810060.0 5170529.0 113444.0 279342.0 7606.0 1712.0 0.0 630213.0 4891187.0 89.0 2.0 4.8 0.1 0.0 0.0 10.8 84.2 100 100 100.00 38 581006000 26.2 23.2 22.8 27.7 0.0 35.9 19.3 smartseq 500172 SRR2049439 SRP059035 SRS951935 SRX1047519 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702687: LC-PT-45-Re_SC08; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702687 GSM1702687 LC-PT-45-Re_SC08 1307317000 6536585 2015-06-05 16:18:03 846273675 1307317000 6536585 2 6536585 index:0,count:6536585,average:100,stdev:0|index:1,count:6536585,average:100,stdev:0 GSM1702687_r1 2.74 2.5 0.07 995064927 1264516531 962116903 1229238774 127.08 127.76 5777180 5168921 252.743 1174.013 153 25727 63.75 66.08 6187991 3683153 6187991 3683153 62.31 62.52 6187991 3599576 6187991 3484442 205461387 20.65 1.67 0 3.11 0 0.14 0 0.07 0 0.00 0 11.41 0 5777180 0 200 0 196.53 0 1.58 0 0.02 0 1.50 0 0.01 0 144.37 0 0.33 0 109438 0 6536585 0 203538 0 8930 0 4634 0 0 0 745841 0 583 0 0 0 10823 0 1623281 0 17834 0 1652521 0 85.27 0 5573642 0 35074 1570979 44.790414552090 6536585.0 5777180.0 109438.0 203538.0 8930.0 4634.0 0.0 745841.0 5573642.0 88.4 1.7 3.1 0.1 0.1 0.0 11.4 85.3 100 100 100.00 38 653658500 27.6 21.7 21.3 29.4 0.0 35.9 18.3 smartseq 500229 SRR2049440 SRP059035 SRS951931 SRX1047520 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702688: LC-PT-45-Re_SC09; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702688 GSM1702688 LC-PT-45-Re_SC09 680301400 3401507 2015-06-05 16:18:03 442125240 680301400 3401507 2 3401507 index:0,count:3401507,average:100,stdev:0|index:1,count:3401507,average:100,stdev:0 GSM1702688_r1 2.71 2.55 0.08 538282163 683342211 517112196 661210813 126.95 127.87 2979259 2616950 274.298 1839.155 175 13473 65.28 68.19 3221246 1944961 3221246 1944961 64.31 64.83 3221246 1916008 3221246 1849237 96956525 18.01 3.12 0 3.73 0 0.18 0 0.02 0 0.00 0 12.21 0 2979259 0 200 0 196.41 0 1.54 0 0.01 0 1.55 0 0.01 0 94.93 0 0.48 0 106213 0 3401507 0 126855 0 6214 0 714 0 0 0 415320 0 384 0 0 0 6989 0 869458 0 7475 0 884306 0 83.86 0 2852404 0 52950 869652 16.424022662890 3401507.0 2979259.0 106213.0 126855.0 6214.0 714.0 0.0 415320.0 2852404.0 87.6 3.1 3.7 0.2 0.0 0.0 12.2 83.9 100 100 100.00 38 340150700 27.2 21.5 21.2 30.0 0.0 35.5 17.3 smartseq 500236 SRR2049441 SRP059035 SRS951930 SRX1047521 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702689: LC-PT-45-Re_SC11; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702689 GSM1702689 LC-PT-45-Re_SC11 1298463400 6492317 2015-06-05 16:18:03 838114317 1298463400 6492317 2 6492317 index:0,count:6492317,average:100,stdev:0|index:1,count:6492317,average:100,stdev:0 GSM1702689_r1 3.29 2.42 0.07 995046578 1276990804 958954990 1238446499 128.33 129.15 5799013 5138607 250.487 1254.986 144 25690 67.55 70.25 6254471 3916985 6254471 3916985 66.24 66.59 6254471 3841250 6254471 3712757 160861216 16.17 1.58 0 3.44 0 0.12 0 0.03 0 0.00 0 10.53 0 5799013 0 200 0 196.49 0 1.51 0 0.02 0 1.54 0 0.01 0 138.30 0 0.33 0 102459 0 6492317 0 223306 0 7578 0 1974 0 0 0 683752 0 1051 0 0 0 13898 0 1782147 0 14264 0 1811360 0 85.88 0 5575707 0 44803 1723816 38.475459232641 6492317.0 5799013.0 102459.0 223306.0 7578.0 1974.0 0.0 683752.0 5575707.0 89.3 1.6 3.4 0.1 0.0 0.0 10.5 85.9 100 100 100.00 38 649231700 27.6 21.8 21.4 29.2 0.0 36.0 18.5 smartseq 500245 SRR2049442 SRP059035 SRS951928 SRX1047522 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702690: LC-PT-45-Re_SC13; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702690 GSM1702690 LC-PT-45-Re_SC13 534738400 2673692 2015-06-05 16:18:03 345307552 534738400 2673692 2 2673692 index:0,count:2673692,average:100,stdev:0|index:1,count:2673692,average:100,stdev:0 GSM1702690_r1 3.94 2.18 0.03 355643319 443378521 338999486 428081500 124.67 126.28 2178066 2030805 225.989 1963.317 90 12538 54.33 57.41 2436884 1183271 2436884 1183271 53.33 54.37 2436884 1161470 2436884 1120541 87191476 24.52 3.68 0 4.38 0 0.37 0 0.05 0 0.00 0 18.12 0 2178066 0 200 0 194.24 0 1.56 0 0.02 0 1.55 0 0.01 0 78.25 0 0.65 0 98493 0 2673692 0 117102 0 10019 0 1236 0 0 0 484371 0 196 0 0 0 4380 0 383116 0 6043 0 393735 0 77.08 0 2060964 0 45984 366191 7.963443806541 2673692.0 2178066.0 98493.0 117102.0 10019.0 1236.0 0.0 484371.0 2060964.0 81.5 3.7 4.4 0.4 0.0 0.0 18.1 77.1 100 100 100.00 38 267369200 26.5 22.0 21.2 30.4 0.0 35.2 16.5 smartseq 500252 SRR2049443 SRP059035 SRS951927 SRX1047523 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702691: LC-PT-45-Re_SC17; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702691 GSM1702691 LC-PT-45-Re_SC17 1293291400 6466457 2015-06-05 16:18:03 841215072 1293291400 6466457 2 6466457 index:0,count:6466457,average:100,stdev:0|index:1,count:6466457,average:100,stdev:0 GSM1702691_r1 2.75 2.76 0.14 997924734 1254778640 964035793 1219315051 125.74 126.48 5794298 5195663 250.029 1182.672 153 25668 61.89 64.18 6223797 3586071 6223797 3586071 60.71 60.86 6223797 3517784 6223797 3400715 198308024 19.87 1.59 0 3.19 0 0.12 0 0.04 0 0.00 0 10.23 0 5794298 0 200 0 196.62 0 1.54 0 0.02 0 1.54 0 0.01 0 129.33 0 0.34 0 102913 0 6466457 0 206479 0 7951 0 2895 0 0 0 661313 0 738 0 0 0 12564 0 1608124 0 14061 0 1635487 0 86.41 0 5587819 0 38581 1555519 40.318265467458 6466457.0 5794298.0 102913.0 206479.0 7951.0 2895.0 0.0 661313.0 5587819.0 89.6 1.6 3.2 0.1 0.0 0.0 10.2 86.4 100 100 100.00 38 646645700 27.8 21.7 21.3 29.2 0.0 36.0 18.8 smartseq 500262 SRR2049444 SRP059035 SRS951929 SRX1047524 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702692: LC-PT-45-Re_SC22; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702692 GSM1702692 LC-PT-45-Re_SC22 835940800 4179704 2015-06-05 16:18:03 546838967 835940800 4179704 2 4179704 index:0,count:4179704,average:100,stdev:0|index:1,count:4179704,average:100,stdev:0 GSM1702692_r1 3.76 2.57 0.1 685768568 859815909 664575068 836610672 125.38 125.89 3771190 3406216 275.214 1388.493 175 16555 58.67 60.67 4011357 2212556 4011357 2212556 57.69 57.84 4011357 2175631 4011357 2109559 162515134 23.70 2.31 0 2.97 0 0.18 0 0.04 0 0.00 0 9.55 0 3771190 0 200 0 196.95 0 1.54 0 0.02 0 1.52 0 0.01 0 146.09 0 0.43 0 96755 0 4179704 0 124253 0 7695 0 1561 0 0 0 399258 0 463 0 0 0 7494 0 855714 0 9072 0 872743 0 87.25 0 3646937 0 43890 860396 19.603463203463 4179704.0 3771190.0 96755.0 124253.0 7695.0 1561.0 0.0 399258.0 3646937.0 90.2 2.3 3.0 0.2 0.0 0.0 9.6 87.3 100 100 100.00 38 417970400 28.1 21.0 20.8 30.1 0.0 36.0 18.3 smartseq 500269 SRR2049445 SRP059035 SRS951926 SRX1047525 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702693: LC-PT-45-Re_SC25; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702693 GSM1702693 LC-PT-45-Re_SC25 1026505600 5132528 2015-06-05 16:18:03 660142693 1026505600 5132528 2 5132528 index:0,count:5132528,average:100,stdev:0|index:1,count:5132528,average:100,stdev:0 GSM1702693_r1 3.91 2.52 0.07 856526204 1069988796 829579516 1040621680 124.92 125.44 4678744 4140332 283.983 1434.707 175 19617 60.77 62.88 4985091 2843124 4985091 2843124 59.8 60.05 4985091 2797719 4985091 2715260 186811827 21.81 2.06 0 3.06 0 0.17 0 0.04 0 0.00 0 8.63 0 4678744 0 200 0 197.21 0 1.56 0 0.02 0 1.51 0 0.01 0 137.89 0 0.37 0 105731 0 5132528 0 157159 0 8818 0 2219 0 0 0 442747 0 779 0 0 0 10173 0 1206657 0 11135 0 1228744 0 88.10 0 4521585 0 48374 1225471 25.333257535039 5132528.0 4678744.0 105731.0 157159.0 8818.0 2219.0 0.0 442747.0 4521585.0 91.2 2.1 3.1 0.2 0.0 0.0 8.6 88.1 100 100 100.00 38 513252800 28.0 21.1 20.9 30.0 0.0 36.0 18.3 smartseq 500277 SRR2049446 SRP059035 SRS951925 SRX1047526 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702694: LC-PT-45-Re_SC26; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702694 GSM1702694 LC-PT-45-Re_SC26 1652227200 8261136 2015-06-05 16:18:03 1063248655 1652227200 8261136 2 8261136 index:0,count:8261136,average:100,stdev:0|index:1,count:8261136,average:100,stdev:0 GSM1702694_r1 3.05 2.53 0.11 1214218696 1537284136 1170062220 1490883012 126.61 127.42 7281632 6512250 239.262 1254.891 133 34066 64.46 67.08 7826544 4693854 7826544 4693854 63.2 63.6 7826544 4602118 7826544 4450494 215757139 17.77 1.83 0 3.44 0 0.16 0 0.03 0 0.00 0 11.66 0 7281632 0 200 0 195.83 0 1.55 0 0.01 0 1.49 0 0.01 0 180.24 0 0.35 0 151058 0 8261136 0 284008 0 13412 0 2728 0 0 0 963364 0 1207 0 0 0 16800 0 2111762 0 18062 0 2147831 0 84.71 0 6997624 0 52545 1991420 37.899324388619 8261136.0 7281632.0 151058.0 284008.0 13412.0 2728.0 0.0 963364.0 6997624.0 88.1 1.8 3.4 0.2 0.0 0.0 11.7 84.7 100 100 100.00 38 826113600 27.7 21.8 21.2 29.3 0.0 36.0 18.8 smartseq 500285 SRR2049447 SRP059035 SRS951923 SRX1047527 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702695: LC-PT-45-Re_SC28; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702695 GSM1702695 LC-PT-45-Re_SC28 1026585000 5132925 2015-06-05 16:18:03 661797046 1026585000 5132925 2 5132925 index:0,count:5132925,average:100,stdev:0|index:1,count:5132925,average:100,stdev:0 GSM1702695_r1 4.62 2.51 0.02 758763480 981203926 729995881 950303562 129.32 130.18 4509317 4029672 236.967 1301.573 145 21236 68.96 71.9 4859605 3109478 4859605 3109478 67.67 68.14 4859605 3051334 4859605 2946684 120337970 15.86 2.01 0 3.60 0 0.18 0 0.04 0 0.00 0 11.94 0 4509317 0 200 0 195.86 0 1.55 0 0.01 0 1.54 0 0.01 0 172.70 0 0.37 0 103278 0 5132925 0 184684 0 9054 0 1896 0 0 0 612658 0 704 0 0 0 10022 0 1338700 0 11249 0 1360675 0 84.25 0 4324633 0 46827 1275952 27.248211501911 5132925.0 4509317.0 103278.0 184684.0 9054.0 1896.0 0.0 612658.0 4324633.0 87.9 2.0 3.6 0.2 0.0 0.0 11.9 84.3 100 100 100.00 38 513292500 27.6 21.6 21.2 29.6 0.0 35.9 18.2 smartseq 500293 SRR2049448 SRP059035 SRS951924 SRX1047528 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702696: LC-PT-45-Re_SC31; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702696 GSM1702696 LC-PT-45-Re_SC31 1154301600 5771508 2015-06-05 16:18:03 746003453 1154301600 5771508 2 5771508 index:0,count:5771508,average:100,stdev:0|index:1,count:5771508,average:100,stdev:0 GSM1702696_r1 3.87 3.2 0.05 978350846 1262359040 943619590 1223940778 129.03 129.71 5329580 4576048 285.653 1492.547 177 21827 69.11 71.77 5709236 3683462 5709236 3683462 67.93 68.15 5709236 3620553 5709236 3497325 153428436 15.68 1.71 0 3.42 0 0.14 0 0.04 0 0.00 0 7.48 0 5329580 0 200 0 197.27 0 1.53 0 0.01 0 1.52 0 0.01 0 136.69 0 0.35 0 98575 0 5771508 0 197593 0 7906 0 2561 0 0 0 431461 0 918 0 0 0 12342 0 1722595 0 12836 0 1748691 0 88.92 0 5131987 0 47813 1755153 36.708698471127 5771508.0 5329580.0 98575.0 197593.0 7906.0 2561.0 0.0 431461.0 5131987.0 92.3 1.7 3.4 0.1 0.0 0.0 7.5 88.9 100 100 100.00 38 577150800 27.8 21.4 21.3 29.4 0.0 36.0 18.8 smartseq 500300 SRR2049449 SRP059035 SRS951922 SRX1047529 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702697: LC-PT-45-Re_SC33; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702697 GSM1702697 LC-PT-45-Re_SC33 846173800 4230869 2015-06-05 16:18:03 548521234 846173800 4230869 2 4230869 index:0,count:4230869,average:100,stdev:0|index:1,count:4230869,average:100,stdev:0 GSM1702697_r1 2.33 3.01 0.07 689411946 868829742 664633382 841648185 126.02 126.63 3791659 3298344 280.472 1653.550 176 16553 65.1 67.71 4058665 2468311 4058665 2468311 64.04 64.4 4058665 2428232 4058665 2347513 122834490 17.82 2.52 0 3.46 0 0.18 0 0.04 0 0.00 0 10.16 0 3791659 0 200 0 196.80 0 1.56 0 0.02 0 1.54 0 0.01 0 120.88 0 0.42 0 106563 0 4230869 0 146237 0 7670 0 1660 0 0 0 429880 0 618 0 0 0 8847 0 1153183 0 11081 0 1173729 0 86.16 0 3645422 0 54267 1158287 21.344223929828 4230869.0 3791659.0 106563.0 146237.0 7670.0 1660.0 0.0 429880.0 3645422.0 89.6 2.5 3.5 0.2 0.0 0.0 10.2 86.2 100 100 100.00 38 423086900 27.6 21.4 21.2 29.9 0.0 35.8 18.0 smartseq 500358 SRR2049450 SRP059035 SRS951921 SRX1047530 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702698: LC-PT-45-Re_SC36; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702698 GSM1702698 LC-PT-45-Re_SC36 1278245200 6391226 2015-06-05 16:18:03 843892096 1278245200 6391226 2 6391226 index:0,count:6391226,average:100,stdev:0|index:1,count:6391226,average:100,stdev:0 GSM1702698_r1 3.27 2.49 0.07 1085841166 1360221499 1046469682 1319687981 125.27 126.11 5900195 5144672 285.338 1390.742 175 24204 64.12 66.63 6321584 3783484 6321584 3783484 62.91 63.22 6321584 3712003 6321584 3589634 199028283 18.33 1.64 0 3.47 0 0.10 0 0.03 0 0.00 0 7.55 0 5900195 0 200 0 197.45 0 1.58 0 0.02 0 1.54 0 0.01 0 167.94 0 0.36 0 104886 0 6391226 0 221942 0 6611 0 2072 0 0 0 482348 0 970 0 0 0 12398 0 1739183 0 13830 0 1766381 0 88.84 0 5678253 0 48443 1754661 36.221146502075 6391226.0 5900195.0 104886.0 221942.0 6611.0 2072.0 0.0 482348.0 5678253.0 92.3 1.6 3.5 0.1 0.0 0.0 7.5 88.8 100 100 100.00 38 639122600 27.9 21.4 21.4 29.3 0.0 36.0 18.8 smartseq 500364 SRR2049451 SRP059035 SRS951920 SRX1047531 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702699: LC-PT-45-Re_SC38; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702699 GSM1702699 LC-PT-45-Re_SC38 1185929600 5929648 2015-06-05 16:18:03 776243425 1185929600 5929648 2 5929648 index:0,count:5929648,average:100,stdev:0|index:1,count:5929648,average:100,stdev:0 GSM1702699_r1 2.04 1.87 0.09 882052289 1134835397 848581993 1098152835 128.66 129.41 5197888 4710249 239.643 1100.577 143 24687 62.72 65.33 5632264 3259973 5632264 3259973 61.44 61.63 5632264 3193760 5632264 3075250 172345611 19.54 1.85 0 3.51 0 0.19 0 0.07 0 0.00 0 12.08 0 5197888 0 200 0 196.07 0 1.57 0 0.02 0 1.48 0 0.01 0 142.31 0 0.36 0 109855 0 5929648 0 208226 0 11203 0 4063 0 0 0 716494 0 723 0 0 0 10323 0 1395638 0 11627 0 1418311 0 84.15 0 4989662 0 29643 1341648 45.260196336403 5929648.0 5197888.0 109855.0 208226.0 11203.0 4063.0 0.0 716494.0 4989662.0 87.7 1.9 3.5 0.2 0.1 0.0 12.1 84.1 100 100 100.00 38 592964800 27.8 21.3 21.0 29.9 0.0 35.9 18.2 smartseq 500372 SRR2049452 SRP059035 SRS951918 SRX1047532 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702700: LC-PT-45-Re_SC39; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702700 GSM1702700 LC-PT-45-Re_SC39 1187801800 5939009 2015-06-05 16:18:03 774704694 1187801800 5939009 2 5939009 index:0,count:5939009,average:100,stdev:0|index:1,count:5939009,average:100,stdev:0 GSM1702700_r1 2.74 3.46 0.08 1012415123 1299399205 976060901 1259988974 128.35 129.09 5490499 4693444 290.239 1470.720 176 22225 71.7 74.49 5882637 3936719 5882637 3936719 70.74 71.11 5882637 3883804 5882637 3757750 139245871 13.75 1.49 0 3.47 0 0.13 0 0.03 0 0.00 0 7.38 0 5490499 0 200 0 197.46 0 1.54 0 0.01 0 1.50 0 0.01 0 146.44 0 0.33 0 88638 0 5939009 0 205824 0 7951 0 2000 0 0 0 438559 0 1086 0 0 0 13125 0 1844094 0 13990 0 1872295 0 88.98 0 5284675 0 49546 1879038 37.925120090421 5939009.0 5490499.0 88638.0 205824.0 7951.0 2000.0 0.0 438559.0 5284675.0 92.4 1.5 3.5 0.1 0.0 0.0 7.4 89.0 100 100 100.00 38 593900900 27.9 21.3 21.3 29.5 0.0 36.0 18.9 smartseq 500380 SRR2049453 SRP059035 SRS951919 SRX1047533 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702701: LC-PT-45-Re_SC40; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702701 GSM1702701 LC-PT-45-Re_SC40 1317261800 6586309 2015-06-05 16:18:03 845415742 1317261800 6586309 2 6586309 index:0,count:6586309,average:100,stdev:0|index:1,count:6586309,average:100,stdev:0 GSM1702701_r1 8.3 2.66 0.09 952735815 1144381957 928395978 1123911380 120.12 121.06 5587193 5438276 244.674 658.980 133 26794 37.1 38.17 5961487 2073028 5961487 2073028 36.54 36.77 5961487 2041458 5961487 1996599 362807887 38.08 1.90 0 2.38 0 0.20 0 0.08 0 0.00 0 14.89 0 5587193 0 200 0 196.64 0 1.53 0 0.02 0 1.54 0 0.01 0 130.28 0 0.32 0 125258 0 6586309 0 156530 0 13054 0 5413 0 0 0 980649 0 336 0 0 0 3593 0 329103 0 11825 0 344857 0 82.45 0 5430663 0 11713 330198 28.190728250662 6586309.0 5587193.0 125258.0 156530.0 13054.0 5413.0 0.0 980649.0 5430663.0 84.8 1.9 2.4 0.2 0.1 0.0 14.9 82.5 100 100 100.00 38 658630900 28.7 20.4 19.9 31.0 0.0 35.9 17.7 smartseq 500388 SRR2049454 SRP059035 SRS951917 SRX1047534 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702702: LC-PT-45-Re_SC43; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702702 GSM1702702 LC-PT-45-Re_SC43 1295288000 6476440 2015-06-05 16:18:03 839799624 1295288000 6476440 2 6476440 index:0,count:6476440,average:100,stdev:0|index:1,count:6476440,average:100,stdev:0 GSM1702702_r1 3.3 2.58 0.07 1095129130 1403616347 1054560182 1359056799 128.17 128.87 5947426 5120744 287.821 1438.769 174 24166 68.13 70.85 6397043 4051820 6397043 4051820 66.99 67.26 6397043 3984346 6397043 3846824 169535738 15.48 1.50 0 3.53 0 0.14 0 0.05 0 0.00 0 7.98 0 5947426 0 200 0 197.39 0 1.52 0 0.01 0 1.54 0 0.01 0 150.42 0 0.35 0 97314 0 6476440 0 228444 0 9280 0 3171 0 0 0 516563 0 1033 0 0 0 14247 0 1916279 0 15724 0 1947283 0 88.30 0 5718982 0 42424 1945137 45.849919856685 6476440.0 5947426.0 97314.0 228444.0 9280.0 3171.0 0.0 516563.0 5718982.0 91.8 1.5 3.5 0.1 0.0 0.0 8.0 88.3 100 100 100.00 38 647644000 27.6 21.7 21.5 29.1 0.0 36.0 18.7 smartseq 500396 SRR2049455 SRP059035 SRS951914 SRX1047535 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702703: LC-PT-45-Re_SC44; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702703 GSM1702703 LC-PT-45-Re_SC44 590297800 2951489 2015-06-05 16:18:03 381431611 590297800 2951489 2 2951489 index:0,count:2951489,average:100,stdev:0|index:1,count:2951489,average:100,stdev:0 GSM1702703_r1 3.57 2.32 0.09 409356256 521918149 390248800 502752473 127.5 128.83 2477759 2255491 230.297 1779.674 152 12426 61.46 64.81 2746934 1522877 2746934 1522877 60.48 61.4 2746934 1498432 2746934 1442729 83830808 20.48 3.26 0 4.34 0 0.26 0 0.04 0 0.00 0 15.76 0 2477759 0 200 0 194.82 0 1.54 0 0.02 0 1.52 0 0.01 0 120.74 0 0.55 0 96079 0 2951489 0 128078 0 7593 0 1038 0 0 0 465099 0 287 0 0 0 5671 0 606016 0 6827 0 618801 0 79.61 0 2349681 0 54960 578286 10.521943231441 2951489.0 2477759.0 96079.0 128078.0 7593.0 1038.0 0.0 465099.0 2349681.0 83.9 3.3 4.3 0.3 0.0 0.0 15.8 79.6 100 100 100.00 38 295148900 26.8 22.1 21.3 29.8 0.0 35.5 17.2 smartseq 500404 SRR2049456 SRP059035 SRS951915 SRX1047536 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702704: LC-PT-45-Re_SC46; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702704 GSM1702704 LC-PT-45-Re_SC46 1463276000 7316380 2015-06-05 16:18:03 940264725 1463276000 7316380 2 7316380 index:0,count:7316380,average:100,stdev:0|index:1,count:7316380,average:100,stdev:0 GSM1702704_r1 8.19 2.43 0.31 1067805869 1261681459 1036844045 1236239394 118.16 119.23 6251095 6056353 244.666 754.459 133 30233 35.78 36.92 6630664 2236875 6630664 2236875 35.23 35.45 6630664 2202345 6630664 2147507 416619405 39.02 1.99 0 2.63 0 0.15 0 0.10 0 0.00 0 14.30 0 6251095 0 200 0 196.62 0 1.59 0 0.02 0 1.46 0 0.01 0 143.15 0 0.34 0 145761 0 7316380 0 192559 0 11242 0 7660 0 0 0 1046383 0 525 0 0 0 7352 0 421054 0 12515 0 441446 0 82.81 0 6058536 0 15419 428040 27.760555159219 7316380.0 6251095.0 145761.0 192559.0 11242.0 7660.0 0.0 1046383.0 6058536.0 85.4 2.0 2.6 0.2 0.1 0.0 14.3 82.8 100 100 100.00 38 731638000 28.5 20.8 20.2 30.5 0.0 35.8 17.7 smartseq 500412 SRR2049457 SRP059035 SRS951913 SRX1047537 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702705: LC-PT-45-Re_SC47; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702705 GSM1702705 LC-PT-45-Re_SC47 939897200 4699486 2015-06-05 16:18:03 619031220 939897200 4699486 2 4699486 index:0,count:4699486,average:100,stdev:0|index:1,count:4699486,average:100,stdev:0 GSM1702705_r1 3.58 3.17 0.07 790924601 1023015244 762329429 991103703 129.34 130.01 4358586 3762228 263.697 1471.747 176 20074 69.73 72.42 4655012 3039125 4655012 3039125 68.17 68.5 4655012 2971163 4655012 2874510 118463727 14.98 1.69 0 3.45 0 0.12 0 0.04 0 0.00 0 7.09 0 4358586 0 200 0 197.05 0 1.57 0 0.01 0 1.53 0 0.01 0 179.98 0 0.39 0 79347 0 4699486 0 162028 0 5495 0 2025 0 0 0 333380 0 912 0 0 0 10550 0 1484185 0 11648 0 1507295 0 89.30 0 4196558 0 46242 1489520 32.211409541110 4699486.0 4358586.0 79347.0 162028.0 5495.0 2025.0 0.0 333380.0 4196558.0 92.7 1.7 3.4 0.1 0.0 0.0 7.1 89.3 100 100 100.00 38 469948600 27.2 22.4 22.3 28.1 0.0 36.1 19.6 smartseq 500421 SRR2049458 SRP059035 SRS951916 SRX1047538 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702706: LC-PT-45-Re_SC51; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702706 GSM1702706 LC-PT-45-Re_SC51 1322010400 6610052 2015-06-05 16:18:03 855032891 1322010400 6610052 2 6610052 index:0,count:6610052,average:100,stdev:0|index:1,count:6610052,average:100,stdev:0 GSM1702706_r1 2.83 2.68 0.07 1121401308 1404650155 1082105709 1359079361 125.26 125.6 6088771 5340027 288.118 1319.428 184 24845 59.78 62.04 6498460 3640028 6498460 3640028 58.98 58.84 6498460 3590893 6498460 3452193 270180773 24.09 1.56 0 3.36 0 0.16 0 0.04 0 0.00 0 7.70 0 6088771 0 200 0 197.51 0 1.53 0 0.01 0 1.51 0 0.01 0 138.35 0 0.33 0 102843 0 6610052 0 221775 0 10265 0 2343 0 0 0 508673 0 942 0 0 0 14016 0 1787589 0 16065 0 1818612 0 88.76 0 5866996 0 41570 1816281 43.692109694491 6610052.0 6088771.0 102843.0 221775.0 10265.0 2343.0 0.0 508673.0 5866996.0 92.1 1.6 3.4 0.2 0.0 0.0 7.7 88.8 100 100 100.00 38 661005200 27.6 21.7 21.6 29.1 0.0 36.0 18.8 smartseq 500429 SRR2049459 SRP059035 SRS951912 SRX1047539 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702707: LC-PT-45-Re_SC53; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702707 GSM1702707 LC-PT-45-Re_SC53 1602442400 8012212 2015-06-05 16:18:03 1033291270 1602442400 8012212 2 8012212 index:0,count:8012212,average:100,stdev:0|index:1,count:8012212,average:100,stdev:0 GSM1702707_r1 4.81 2.34 0.12 1211639910 1412619578 1178705286 1384412478 116.59 117.45 7042666 6704540 249.197 888.576 143 32267 39.71 40.89 7450169 2796957 7450169 2796957 38.95 39.2 7450169 2743331 7450169 2681739 385780086 31.84 1.69 0 2.52 0 0.16 0 0.09 0 0.00 0 11.85 0 7042666 0 200 0 196.95 0 1.59 0 0.02 0 1.53 0 0.01 0 183.72 0 0.34 0 135056 0 8012212 0 202055 0 12639 0 7074 0 0 0 949833 0 81 0 0 0 9080 0 765451 0 15234 0 789846 0 85.38 0 6840611 0 24511 751472 30.658561462201 8012212.0 7042666.0 135056.0 202055.0 12639.0 7074.0 0.0 949833.0 6840611.0 87.9 1.7 2.5 0.2 0.1 0.0 11.9 85.4 100 100 100.00 38 801221200 28.5 21.0 20.5 30.1 0.0 36.0 18.3 smartseq 500485 SRR2049460 SRP059035 SRS951911 SRX1047540 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702708: LC-PT-45-Re_SC54; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702708 GSM1702708 LC-PT-45-Re_SC54 674509400 3372547 2015-06-05 16:18:03 443038911 674509400 3372547 2 3372547 index:0,count:3372547,average:100,stdev:0|index:1,count:3372547,average:100,stdev:0 GSM1702708_r1 4.41 2.54 0.09 491415174 644137047 470989714 621088907 131.08 131.87 2928103 2592050 236.335 1470.585 153 14191 72.45 75.87 3165473 2121328 3165473 2121328 71.11 71.68 3165473 2082122 3165473 2004279 70292351 14.30 2.09 0 3.92 0 0.15 0 0.03 0 0.00 0 13.00 0 2928103 0 200 0 195.64 0 1.54 0 0.01 0 1.51 0 0.01 0 126.47 0 0.46 0 70440 0 3372547 0 132129 0 5151 0 850 0 0 0 438443 0 516 0 0 0 7619 0 992900 0 8818 0 1009853 0 82.90 0 2795974 0 54667 941653 17.225254724057 3372547.0 2928103.0 70440.0 132129.0 5151.0 850.0 0.0 438443.0 2795974.0 86.8 2.1 3.9 0.2 0.0 0.0 13.0 82.9 100 100 100.00 38 337254700 27.0 22.1 21.7 29.2 0.0 35.7 17.9 smartseq 500493 SRR2049461 SRP059035 SRS951909 SRX1047541 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702709: LC-PT-45-Re_SC55; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702709 GSM1702709 LC-PT-45-Re_SC55 808599600 4042998 2015-06-05 16:18:03 521321037 808599600 4042998 2 4042998 index:0,count:4042998,average:100,stdev:0|index:1,count:4042998,average:100,stdev:0 GSM1702709_r1 4.19 2.2 0.07 654839076 844455915 629805573 817801839 128.96 129.85 3600798 3128477 281.344 2002.076 175 15321 69.72 72.73 3909087 2510530 3909087 2510530 68.65 69.25 3909087 2472015 3909087 2390545 94725154 14.47 2.54 0 3.68 0 0.25 0 0.15 0 0.00 0 10.54 0 3600798 0 200 0 196.77 0 1.54 0 0.02 0 1.54 0 0.01 0 126.56 0 0.43 0 102556 0 4042998 0 148746 0 9989 0 6135 0 0 0 426076 0 509 0 0 0 9160 0 1083799 0 9661 0 1103129 0 85.38 0 3452052 0 57237 1096404 19.155511295141 4042998.0 3600798.0 102556.0 148746.0 9989.0 6135.0 0.0 426076.0 3452052.0 89.1 2.5 3.7 0.2 0.2 0.0 10.5 85.4 100 100 100.00 38 404299800 27.3 21.6 21.3 29.9 0.0 35.7 17.8 smartseq 500502 SRR2049462 SRP059035 SRS951910 SRX1047542 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702710: LC-PT-45-Re_SC56; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702710 GSM1702710 LC-PT-45-Re_SC56 1112741000 5563705 2015-06-05 16:18:03 716759995 1112741000 5563705 2 5563705 index:0,count:5563705,average:100,stdev:0|index:1,count:5563705,average:100,stdev:0 GSM1702710_r1 3.3 2.65 0.09 928474226 1178480410 894538150 1142799625 126.93 127.75 5063264 4448885 285.473 1460.671 175 21079 64.35 66.94 5467331 3258433 5467331 3258433 63.38 63.75 5467331 3208986 5467331 3103158 174542341 18.80 1.85 0 3.52 0 0.17 0 0.05 0 0.00 0 8.77 0 5063264 0 200 0 197.26 0 1.55 0 0.02 0 1.53 0 0.01 0 133.53 0 0.36 0 102915 0 5563705 0 195832 0 9716 0 2714 0 0 0 488011 0 746 0 0 0 10977 0 1424937 0 12118 0 1448778 0 87.49 0 4867432 0 45670 1440623 31.544186555726 5563705.0 5063264.0 102915.0 195832.0 9716.0 2714.0 0.0 488011.0 4867432.0 91.0 1.8 3.5 0.2 0.0 0.0 8.8 87.5 100 100 100.00 38 556370500 27.8 21.3 21.1 29.8 0.0 35.9 18.2 smartseq 500509 SRR2049463 SRP059035 SRS951908 SRX1047543 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702711: LC-PT-45-Re_SC57; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702711 GSM1702711 LC-PT-45-Re_SC57 1225642800 6128214 2015-06-05 16:18:03 789146260 1225642800 6128214 2 6128214 index:0,count:6128214,average:100,stdev:0|index:1,count:6128214,average:100,stdev:0 GSM1702711_r1 2.48 2.6 0.08 873339753 1099763818 839161612 1064183771 125.93 126.82 5286434 4749706 235.349 1415.624 133 25303 64.06 66.92 5741660 3386385 5741660 3386385 62.82 63.37 5741660 3320996 5741660 3206789 162915792 18.65 2.11 0 3.69 0 0.17 0 0.03 0 0.00 0 13.53 0 5286434 0 200 0 195.41 0 1.56 0 0.02 0 1.53 0 0.01 0 118.61 0 0.41 0 129061 0 6128214 0 226310 0 10634 0 1932 0 0 0 829214 0 608 0 0 0 12444 0 1491664 0 13353 0 1518069 0 82.57 0 5060124 0 57423 1405050 24.468418577922 6128214.0 5286434.0 129061.0 226310.0 10634.0 1932.0 0.0 829214.0 5060124.0 86.3 2.1 3.7 0.2 0.0 0.0 13.5 82.6 100 100 100.00 38 612821400 27.4 21.8 21.1 29.7 0.0 35.8 18.0 smartseq 500517 SRR2049464 SRP059035 SRS951907 SRX1047544 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702712: LC-PT-45-Re_SC59; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702712 GSM1702712 LC-PT-45-Re_SC59 1146898200 5734491 2015-06-05 16:18:03 747000814 1146898200 5734491 2 5734491 index:0,count:5734491,average:100,stdev:0|index:1,count:5734491,average:100,stdev:0 GSM1702712_r1 3.45 2.94 0.01 838686682 1094615754 789002563 1040830868 130.52 131.92 4904688 4197869 248.320 2135.774 153 21944 75.97 81.06 5628955 3726285 5628955 3726285 75.75 76.35 5628955 3715131 5628955 3509792 78698572 9.38 1.75 0 5.36 0 0.15 0 0.01 0 0.00 0 14.30 0 4904688 0 200 0 195.93 0 1.60 0 0.02 0 1.50 0 0.01 0 112.81 0 0.34 0 100307 0 5734491 0 307563 0 8743 0 797 0 0 0 820263 0 547 0 0 0 13872 0 1949248 0 15595 0 1979262 0 80.17 0 4597125 0 26048 1908411 73.265164312039 5734491.0 4904688.0 100307.0 307563.0 8743.0 797.0 0.0 820263.0 4597125.0 85.5 1.7 5.4 0.2 0.0 0.0 14.3 80.2 100 100 100.00 38 573449100 26.7 22.4 22.2 28.8 0.0 35.6 17.9 smartseq 500525 SRR2049465 SRP059035 SRS951906 SRX1047545 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702713: LC-PT-45-Re_SC61; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702713 GSM1702713 LC-PT-45-Re_SC61 1794754600 8973773 2015-06-05 16:18:03 1154995143 1794754600 8973773 2 8973773 index:0,count:8973773,average:100,stdev:0|index:1,count:8973773,average:100,stdev:0 GSM1702713_r1 3.56 2.85 0.1 1318398687 1668682550 1271051479 1616788259 126.57 127.2 7875286 7011387 240.722 1228.558 143 35723 66.64 69.33 8483159 5248328 8483159 5248328 65.47 65.82 8483159 5156210 8483159 4982720 218976887 16.61 1.71 0 3.40 0 0.15 0 0.03 0 0.00 0 12.07 0 7875286 0 200 0 195.86 0 1.57 0 0.02 0 1.50 0 0.01 0 148.87 0 0.34 0 153277 0 8973773 0 305268 0 13369 0 2413 0 0 0 1082705 0 1414 0 0 0 17437 0 2377147 0 20038 0 2416036 0 84.36 0 7570018 0 53998 2240945 41.500518537724 8973773.0 7875286.0 153277.0 305268.0 13369.0 2413.0 0.0 1082705.0 7570018.0 87.8 1.7 3.4 0.1 0.0 0.0 12.1 84.4 100 100 100.00 38 897377300 27.5 21.8 21.3 29.4 0.0 35.9 18.3 smartseq 500533 SRR2049466 SRP059035 SRS951905 SRX1047546 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702714: LC-PT-45-Re_SC67; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702714 GSM1702714 LC-PT-45-Re_SC67 1479954600 7399773 2015-06-05 16:18:03 950732937 1479954600 7399773 2 7399773 index:0,count:7399773,average:100,stdev:0|index:1,count:7399773,average:100,stdev:0 GSM1702714_r1 6.45 2.52 0.08 1122268767 1300274941 1090045174 1273209009 115.86 116.8 6553007 6342459 242.038 732.105 133 31000 33.36 34.4 6949787 2186221 6949787 2186221 32.98 33.14 6949787 2161020 6949787 2106169 440080625 39.21 1.92 0 2.66 0 0.15 0 0.08 0 0.00 0 11.22 0 6553007 0 200 0 196.82 0 1.61 0 0.02 0 1.49 0 0.01 0 153.98 0 0.32 0 142285 0 7399773 0 197161 0 10929 0 5804 0 0 0 830033 0 341 0 0 0 6565 0 461368 0 15525 0 483799 0 85.89 0 6355846 0 19807 488682 24.672186600697 7399773.0 6553007.0 142285.0 197161.0 10929.0 5804.0 0.0 830033.0 6355846.0 88.6 1.9 2.7 0.1 0.1 0.0 11.2 85.9 100 100 100.00 38 739977300 28.7 20.8 20.2 30.3 0.0 36.1 18.5 smartseq 500541 SRR2049467 SRP059035 SRS951904 SRX1047547 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702715: LC-PT-45-Re_SC69; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702715 GSM1702715 LC-PT-45-Re_SC69 1182224200 5911121 2015-06-05 16:18:03 769297929 1182224200 5911121 2 5911121 index:0,count:5911121,average:100,stdev:0|index:1,count:5911121,average:100,stdev:0 GSM1702715_r1 5.35 2.22 0.1 905345195 1149703152 870316400 1112467609 126.99 127.82 5272162 4746404 251.074 1123.476 153 23426 61.94 64.59 5698987 3265581 5698987 3265581 60.93 61.25 5698987 3212275 5698987 3096885 183403797 20.26 1.60 0 3.66 0 0.25 0 0.06 0 0.00 0 10.50 0 5272162 0 200 0 196.52 0 1.55 0 0.02 0 1.53 0 0.01 0 125.92 0 0.35 0 94661 0 5911121 0 216392 0 14798 0 3464 0 0 0 620697 0 646 0 0 0 11090 0 1412889 0 12402 0 1437027 0 85.53 0 5055770 0 41961 1372594 32.711184194848 5911121.0 5272162.0 94661.0 216392.0 14798.0 3464.0 0.0 620697.0 5055770.0 89.2 1.6 3.7 0.3 0.1 0.0 10.5 85.5 100 100 100.00 38 591112100 27.8 21.5 21.2 29.5 0.0 36.0 18.5 smartseq 500549 SRR2049468 SRP059035 SRS951903 SRX1047548 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702716: LC-PT-45-Re_SC70; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702716 GSM1702716 LC-PT-45-Re_SC70 1103646800 5518234 2015-06-05 16:18:03 714012512 1103646800 5518234 2 5518234 index:0,count:5518234,average:100,stdev:0|index:1,count:5518234,average:100,stdev:0 GSM1702716_r1 3.51 2.65 0.07 923598768 1203357895 888522050 1164915728 130.29 131.11 5046133 4266520 285.383 1565.805 176 20846 75.69 78.94 5461189 3819497 5461189 3819497 74.41 75.08 5461189 3755053 5461189 3632668 105966820 11.47 1.66 0 3.77 0 0.23 0 0.04 0 0.00 0 8.29 0 5046133 0 200 0 197.23 0 1.55 0 0.01 0 1.57 0 0.01 0 121.88 0 0.35 0 91527 0 5518234 0 207815 0 12587 0 2212 0 0 0 457302 0 700 0 0 0 12790 0 1808017 0 13647 0 1835154 0 87.68 0 4838318 0 52244 1827428 34.978715259169 5518234.0 5046133.0 91527.0 207815.0 12587.0 2212.0 0.0 457302.0 4838318.0 91.4 1.7 3.8 0.2 0.0 0.0 8.3 87.7 100 100 100.00 38 551823400 27.6 21.4 21.4 29.6 0.0 36.0 18.6 smartseq 500557 SRR2049469 SRP059035 SRS951902 SRX1047549 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702717: LC-PT-45-Re_SC71; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702717 GSM1702717 LC-PT-45-Re_SC71 973909800 4869549 2015-06-05 16:18:03 629573909 973909800 4869549 2 4869549 index:0,count:4869549,average:100,stdev:0|index:1,count:4869549,average:100,stdev:0 GSM1702717_r1 3.81 2.66 0.09 719357499 902890459 692873537 874457755 125.51 126.21 4269466 3892155 240.248 1310.123 144 20333 58.77 61.21 4599455 2509041 4599455 2509041 57.74 58.02 4599455 2465091 4599455 2378224 162974328 22.66 2.24 0 3.51 0 0.18 0 0.05 0 0.00 0 12.09 0 4269466 0 200 0 195.85 0 1.57 0 0.02 0 1.51 0 0.01 0 118.45 0 0.41 0 109304 0 4869549 0 170681 0 8982 0 2470 0 0 0 588631 0 591 0 0 0 9010 0 1040738 0 12169 0 1062508 0 84.17 0 4098785 0 47665 996272 20.901542011958 4869549.0 4269466.0 109304.0 170681.0 8982.0 2470.0 0.0 588631.0 4098785.0 87.7 2.2 3.5 0.2 0.1 0.0 12.1 84.2 100 100 100.00 38 486954900 27.5 21.8 21.2 29.5 0.0 35.8 18.2 smartseq 500613 SRR2049470 SRP059035 SRS951901 SRX1047550 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702718: LC-PT-45-Re_SC72; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702718 GSM1702718 LC-PT-45-Re_SC72 1493094600 7465473 2015-06-05 16:18:03 961208691 1493094600 7465473 2 7465473 index:0,count:7465473,average:100,stdev:0|index:1,count:7465473,average:100,stdev:0 GSM1702718_r1 4.58 2.59 0.06 1155157614 1446462900 1115995290 1405213060 125.22 125.92 6717174 6025575 247.713 1142.786 155 30075 62.4 64.71 7185118 4191694 7185118 4191694 61.7 61.85 7185118 4144279 7185118 4006607 220825787 19.12 1.52 0 3.21 0 0.13 0 0.04 0 0.00 0 9.85 0 6717174 0 200 0 196.68 0 1.54 0 0.02 0 1.49 0 0.01 0 182.83 0 0.32 0 113812 0 7465473 0 239578 0 9697 0 2931 0 0 0 735671 0 927 0 0 0 14301 0 1782003 0 16704 0 1813935 0 86.77 0 6477596 0 40254 1743585 43.314577433299 7465473.0 6717174.0 113812.0 239578.0 9697.0 2931.0 0.0 735671.0 6477596.0 90.0 1.5 3.2 0.1 0.0 0.0 9.9 86.8 100 100 100.00 38 746547300 27.9 21.6 21.2 29.3 0.0 36.1 18.9 smartseq 500628 SRR2049472 SRP059035 SRS951899 SRX1047552 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702720: LC-PT-45-Re_SC80; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702720 GSM1702720 LC-PT-45-Re_SC80 1297756600 6488783 2015-06-05 16:18:03 839250123 1297756600 6488783 2 6488783 index:0,count:6488783,average:100,stdev:0|index:1,count:6488783,average:100,stdev:0 GSM1702720_r1 3.52 2.76 0.1 1101375941 1382373389 1065036257 1343193257 125.51 126.12 5995063 5220740 286.016 1453.079 178 24747 63.11 65.36 6382643 3783367 6382643 3783367 61.83 62.07 6382643 3706781 6382643 3592592 229274202 20.82 1.79 0 3.19 0 0.13 0 0.05 0 0.00 0 7.43 0 5995063 0 200 0 197.37 0 1.56 0 0.02 0 1.53 0 0.01 0 151.69 0 0.35 0 116464 0 6488783 0 206676 0 8581 0 3174 0 0 0 481965 0 1062 0 0 0 13268 0 1776124 0 16318 0 1806772 0 89.21 0 5788387 0 52269 1796775 34.375538081846 6488783.0 5995063.0 116464.0 206676.0 8581.0 3174.0 0.0 481965.0 5788387.0 92.4 1.8 3.2 0.1 0.0 0.0 7.4 89.2 100 100 100.00 38 648878300 27.9 21.5 21.4 29.3 0.0 36.1 18.9 smartseq 500676 SRR2049478 SRP059035 SRS951896 SRX1047558 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702726: LC-PT-45-Re_SC91; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702726 GSM1702726 LC-PT-45-Re_SC91 833771600 4168858 2015-06-05 16:18:03 542309333 833771600 4168858 2 4168858 index:0,count:4168858,average:100,stdev:0|index:1,count:4168858,average:100,stdev:0 GSM1702726_r1 3.07 2.62 0.08 684900322 859891857 661510078 835942135 125.55 126.37 3753400 3314933 283.295 1680.869 176 15971 61.44 63.77 4012083 2305968 4012083 2305968 60.4 60.76 4012083 2267093 4012083 2197166 146833049 21.44 2.55 0 3.29 0 0.16 0 0.04 0 0.00 0 9.77 0 3753400 0 200 0 196.88 0 1.56 0 0.02 0 1.55 0 0.01 0 154.72 0 0.43 0 106135 0 4168858 0 137168 0 6585 0 1624 0 0 0 407249 0 512 0 0 0 7805 0 1006387 0 9672 0 1024376 0 86.74 0 3616232 0 49933 1013383 20.294855105842 4168858.0 3753400.0 106135.0 137168.0 6585.0 1624.0 0.0 407249.0 3616232.0 90.0 2.5 3.3 0.2 0.0 0.0 9.8 86.7 100 100 100.00 38 416885800 27.7 21.4 21.2 29.8 0.0 35.8 18.1 smartseq 500684 SRR2049479 SRP059035 SRS951891 SRX1047559 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702727: LC-PT-45-Re_SC92; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45-Re GEO Accession;;GSM1702727 GSM1702727 LC-PT-45-Re_SC92 896374600 4481873 2015-06-05 16:18:03 576389402 896374600 4481873 2 4481873 index:0,count:4481873,average:100,stdev:0|index:1,count:4481873,average:100,stdev:0 GSM1702727_r1 2.18 2.3 0.06 740569671 931091090 714141405 903355790 125.73 126.5 4055065 3554653 283.595 1621.481 175 17207 63.63 66.14 4355622 2580067 4355622 2580067 62.67 63.04 4355622 2541243 4355622 2459404 142836451 19.29 2.39 0 3.43 0 0.16 0 0.04 0 0.00 0 9.32 0 4055065 0 200 0 196.97 0 1.55 0 0.02 0 1.54 0 0.01 0 118.64 0 0.40 0 107074 0 4481873 0 153853 0 7146 0 1741 0 0 0 417921 0 720 0 0 0 9367 0 1168622 0 10629 0 1189338 0 87.04 0 3901212 0 53034 1180919 22.267205943357 4481873.0 4055065.0 107074.0 153853.0 7146.0 1741.0 0.0 417921.0 3901212.0 90.5 2.4 3.4 0.2 0.0 0.0 9.3 87.0 100 100 100.00 38 448187300 27.7 21.4 21.2 29.7 0.0 35.9 18.2 smartseq 500740 SRR2049480 SRP059035 SRS951892 SRX1047560 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702728: LC-MBT-15_Afatinib; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702728 GSM1702728 LC-MBT-15_Afatinib 2066083800 10330419 2015-06-05 16:18:03 1364643280 2066083800 10330419 2 10330419 index:0,count:10330419,average:100,stdev:0|index:1,count:10330419,average:100,stdev:0 GSM1702728_r1 2.9 4.25 0.01 1727192211 2341004640 1664185530 2269930769 135.54 136.4 9480534 6832687 327.862 2645.366 174 23798 89.64 93.21 10314414 8498222 10314414 8498222 87.87 88.61 10314414 8330298 10314414 8078759 48842072 2.83 1.28 0 3.52 0 0.07 0 0.03 0 0.00 0 8.13 0 9480534 0 200 0 196.70 0 1.57 0 0.01 0 1.51 0 0.01 0 222.69 0 0.38 0 132621 0 10330419 0 363325 0 7665 0 2597 0 0 0 839623 0 1962 0 0 0 39643 0 4712075 0 24122 0 4777802 0 88.26 0 9117209 0 140790 4650263 33.029781944740 10330419.0 9480534.0 132621.0 363325.0 7665.0 2597.0 0.0 839623.0 9117209.0 91.8 1.3 3.5 0.1 0.0 0.0 8.1 88.3 100 100 100.00 38 1033041900 26.3 23.3 23.2 27.2 0.0 36.0 20.0 smartseq 500748 SRR2049481 SRP059035 SRS951890 SRX1047561 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702729: LC-MBT-15_Carboplatin; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702729 GSM1702729 LC-MBT-15_Carboplatin 1122156600 5610783 2015-06-05 16:18:03 741321328 1122156600 5610783 2 5610783 index:0,count:5610783,average:100,stdev:0|index:1,count:5610783,average:100,stdev:0 GSM1702729_r1 4.11 4.11 0.01 885852623 1199144042 846674772 1155268416 135.37 136.45 4921849 3658391 325.315 2874.319 162 13181 85.98 90.25 5478166 4231956 5478166 4231956 84.9 85.96 5478166 4178755 5478166 4030850 37705206 4.26 3.00 0 4.15 0 0.11 0 0.03 0 0.00 0 12.14 0 4921849 0 200 0 195.80 0 1.55 0 0.01 0 1.53 0 0.01 0 124.68 0 0.51 0 168600 0 5610783 0 232705 0 6151 0 1596 0 0 0 681187 0 844 0 0 0 17154 0 2172897 0 14746 0 2205641 0 83.57 0 4689144 0 114382 2152124 18.815233166058 5610783.0 4921849.0 168600.0 232705.0 6151.0 1596.0 0.0 681187.0 4689144.0 87.7 3.0 4.1 0.1 0.0 0.0 12.1 83.6 100 100 100.00 38 561078300 26.1 23.2 23.0 27.7 0.0 35.7 19.0 smartseq 500757 SRR2049482 SRP059035 SRS951889 SRX1047562 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702730: LC-MBT-15_DAPT; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702730 GSM1702730 LC-MBT-15_DAPT 1929574200 9647871 2015-06-05 16:18:03 1273707146 1929574200 9647871 2 9647871 index:0,count:9647871,average:100,stdev:0|index:1,count:9647871,average:100,stdev:0 GSM1702730_r1 4.13 4.19 0.01 1565899383 2119902474 1503624073 2050007641 135.38 136.34 8666719 6436699 323.039 2632.605 162 22571 87.07 90.92 9513289 7546315 9513289 7546315 85.88 86.77 9513289 7443180 9513289 7201831 62274349 3.98 2.13 0 3.80 0 0.09 0 0.03 0 0.00 0 10.05 0 8666719 0 200 0 196.31 0 1.55 0 0.01 0 1.51 0 0.01 0 162.30 0 0.43 0 205863 0 9647871 0 366446 0 8971 0 2906 0 0 0 969275 0 1656 0 0 0 28008 0 3953459 0 23692 0 4006815 0 86.03 0 8300273 0 135172 3899757 28.850331429586 9647871.0 8666719.0 205863.0 366446.0 8971.0 2906.0 0.0 969275.0 8300273.0 89.8 2.1 3.8 0.1 0.0 0.0 10.0 86.0 100 100 100.00 38 964787100 26.5 23.0 22.9 27.6 0.0 35.9 19.5 smartseq 500765 SRR2049483 SRP059035 SRS951888 SRX1047563 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702731: LC-MBT-15_Docetaxel; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702731 GSM1702731 LC-MBT-15_Docetaxel 1451136400 7255682 2015-06-05 16:18:03 962426997 1451136400 7255682 2 7255682 index:0,count:7255682,average:100,stdev:0|index:1,count:7255682,average:100,stdev:0 GSM1702731_r1 3.69 4.16 0.01 1168949980 1580150514 1120473157 1525784516 135.18 136.17 6463745 4781225 326.051 2698.979 165 16766 87.69 91.75 7138141 5668111 7138141 5668111 86.55 87.53 7138141 5594058 7138141 5407261 40554331 3.47 2.13 0 3.94 0 0.11 0 0.03 0 0.00 0 10.78 0 6463745 0 200 0 196.27 0 1.57 0 0.01 0 1.51 0 0.01 0 152.75 0 0.45 0 154750 0 7255682 0 286160 0 7874 0 2168 0 0 0 781895 0 1264 0 0 0 21254 0 2957292 0 17204 0 2997014 0 85.14 0 6177585 0 124582 2925801 23.484941644860 7255682.0 6463745.0 154750.0 286160.0 7874.0 2168.0 0.0 781895.0 6177585.0 89.1 2.1 3.9 0.1 0.0 0.0 10.8 85.1 100 100 100.00 38 725568200 26.4 23.0 22.7 27.9 0.0 35.7 19.1 smartseq 500773 SRR2049484 SRP059035 SRS951887 SRX1047564 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702732: LC-MBT-15_Erlotinib; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702732 GSM1702732 LC-MBT-15_Erlotinib 1652241000 8261205 2015-06-05 16:18:03 1085840847 1652241000 8261205 2 8261205 index:0,count:8261205,average:100,stdev:0|index:1,count:8261205,average:100,stdev:0 GSM1702732_r1 3.82 4.18 0.01 1342258768 1815697564 1287793576 1755102486 135.27 136.29 7450056 5528541 316.100 2646.518 160 19909 87.28 91.18 8209500 6502282 8209500 6502282 85.92 86.81 8209500 6401116 8209500 6190953 51533735 3.84 1.94 0 3.86 0 0.08 0 0.03 0 0.00 0 9.71 0 7450056 0 200 0 196.32 0 1.54 0 0.01 0 1.51 0 0.01 0 164.31 0 0.43 0 160575 0 8261205 0 318778 0 6945 0 2445 0 0 0 801759 0 1545 0 0 0 28621 0 3465978 0 20465 0 3516609 0 86.32 0 7131278 0 131627 3416252 25.954036785766 8261205.0 7450056.0 160575.0 318778.0 6945.0 2445.0 0.0 801759.0 7131278.0 90.2 1.9 3.9 0.1 0.0 0.0 9.7 86.3 100 100 100.00 38 826120500 26.3 23.3 23.1 27.3 0.0 35.9 19.6 smartseq 500781 SRR2049485 SRP059035 SRS951886 SRX1047565 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702733: LC-MBT-15_Tivantinib; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702733 GSM1702733 LC-MBT-15_Tivantinib 2004324000 10021620 2015-06-05 16:18:03 1400509228 2004324000 10021620 2 10021620 index:0,count:10021620,average:100,stdev:0|index:1,count:10021620,average:100,stdev:0 GSM1702733_r1 5.66 3.65 0.03 1254409345 1742129448 1167663876 1635939966 138.88 140.1 7759105 6755190 237.612 2464.880 90 46676 78.31 84.7 9030355 6076003 9030355 6076003 78.32 79.93 9030355 6076676 9030355 5734281 81747166 6.52 3.27 0 5.84 0 0.19 0 0.02 0 0.00 0 22.37 0 7759105 0 200 0 192.77 0 1.51 0 0.01 0 1.54 0 0.01 0 96.72 0 0.94 0 327626 0 10021620 0 585197 0 19064 0 1933 0 0 0 2241518 0 721 0 0 0 24971 0 2626692 0 21923 0 2674307 0 71.58 0 7173908 0 109034 2541023 23.304868206248 10021620.0 7759105.0 327626.0 585197.0 19064.0 1933.0 0.0 2241518.0 7173908.0 77.4 3.3 5.8 0.2 0.0 0.0 22.4 71.6 100 100 100.00 38 1002162000 24.5 24.8 23.5 27.2 0.0 34.1 16.7 smartseq 500789 SRR2049486 SRP059035 SRS951885 SRX1047566 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702734: LC-MBT-15_Pooled; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702734 GSM1702734 LC-MBT-15_Pooled 1974479800 9872399 2015-06-05 16:18:03 1395555055 1974479800 9872399 2 9872399 index:0,count:9872399,average:100,stdev:0|index:1,count:9872399,average:100,stdev:0 GSM1702734_r1 1.9 4.41 0.02 1372575195 1885790430 1293161559 1784892788 137.39 138.03 8235261 6747016 250.737 1900.024 162 32165 88.82 94.7 9179928 7314176 9179928 7314176 88.13 89.15 9179928 7257497 9179928 6885677 27268916 1.99 2.03 0 5.18 0 0.09 0 0.01 0 0.00 0 16.48 0 8235261 0 200 0 194.41 0 1.57 0 0.01 0 1.51 0 0.01 0 121.71 0 0.64 0 200555 0 9872399 0 511834 0 8643 0 1226 0 0 0 1627269 0 1304 0 0 0 25257 0 3948327 0 20131 0 3995019 0 78.23 0 7723427 0 115843 3752546 32.393377243338 9872399.0 8235261.0 200555.0 511834.0 8643.0 1226.0 0.0 1627269.0 7723427.0 83.4 2.0 5.2 0.1 0.0 0.0 16.5 78.2 100 100 100.00 38 987239900 25.6 24.0 23.2 27.2 0.0 34.9 18.4 smartseq 500798 SRR2049487 SRP059035 SRS951884 SRX1047567 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702735: LC-MBT-15_SC02; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702735 GSM1702735 LC-MBT-15_SC02 1321905800 6609529 2015-06-05 16:18:03 874792798 1321905800 6609529 2 6609529 index:0,count:6609529,average:100,stdev:0|index:1,count:6609529,average:100,stdev:0 GSM1702735_r1 2.41 3.97 0.02 904506973 1132691598 873163882 1096161459 125.23 125.54 5564657 5009145 230.000 1230.343 111 26395 61.71 64.14 5946226 3433957 5946226 3433957 60.02 60.03 5946226 3339671 5946226 3214048 178080773 19.69 1.55 0 3.19 0 0.09 0 0.03 0 0.00 0 15.69 0 5564657 0 200 0 194.86 0 1.56 0 0.02 0 1.46 0 0.01 0 164.10 0 0.39 0 102270 0 6609529 0 210623 0 5685 0 2145 0 0 0 1037042 0 604 0 0 0 11386 0 1531803 0 13265 0 1557058 0 81.00 0 5354034 0 49464 1422531 28.758915574964 6609529.0 5564657.0 102270.0 210623.0 5685.0 2145.0 0.0 1037042.0 5354034.0 84.2 1.5 3.2 0.1 0.0 0.0 15.7 81.0 100 100 100.00 38 660952900 27.3 22.3 21.3 29.1 0.0 35.7 18.2 smartseq 500900 SRR2049494 SRP059035 SRS951877 SRX1047574 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702742: LC-MBT-15_SC15; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702742 GSM1702742 LC-MBT-15_SC15 1348262200 6741311 2015-06-05 16:18:03 889399780 1348262200 6741311 2 6741311 index:0,count:6741311,average:100,stdev:0|index:1,count:6741311,average:100,stdev:0 GSM1702742_r1 2.33 4.03 0.01 1014593094 1281310665 980916389 1242252189 126.29 126.64 6005782 5343987 242.508 1383.287 142 27517 65.88 68.33 6386162 3956315 6386162 3956315 64.49 64.71 6386162 3873359 6386162 3746632 178337265 17.58 1.90 0 3.21 0 0.11 0 0.03 0 0.00 0 10.77 0 6005782 0 200 0 195.97 0 1.57 0 0.02 0 1.46 0 0.01 0 141.10 0 0.43 0 128099 0 6741311 0 216167 0 7205 0 2133 0 0 0 726191 0 863 0 0 0 14625 0 1767016 0 14832 0 1797336 0 85.88 0 5789615 0 59319 1680396 28.328124209781 6741311.0 6005782.0 128099.0 216167.0 7205.0 2133.0 0.0 726191.0 5789615.0 89.1 1.9 3.2 0.1 0.0 0.0 10.8 85.9 100 100 100.00 38 674131100 27.6 21.7 21.3 29.5 0.0 35.8 18.3 smartseq 500908 SRR2049495 SRP059035 SRS951876 SRX1047575 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702743: LC-MBT-15_SC16; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702743 GSM1702743 LC-MBT-15_SC16 1365834400 6829172 2015-06-05 16:18:03 901776433 1365834400 6829172 2 6829172 index:0,count:6829172,average:100,stdev:0|index:1,count:6829172,average:100,stdev:0 GSM1702743_r1 3.37 3.97 0.02 903036938 1141448506 871324152 1105244845 126.4 126.85 5632363 5060161 225.648 1376.337 111 28758 65.21 67.85 6010076 3672847 6010076 3672847 63.84 64.11 6010076 3595594 6010076 3470396 156061903 17.28 1.84 0 3.20 0 0.11 0 0.04 0 0.00 0 17.38 0 5632363 0 200 0 194.25 0 1.58 0 0.02 0 1.48 0 0.01 0 108.30 0 0.44 0 125661 0 6829172 0 218819 0 7173 0 2531 0 0 0 1187105 0 652 0 0 0 12399 0 1567232 0 15504 0 1595787 0 79.27 0 5413544 0 57202 1445898 25.277053249886 6829172.0 5632363.0 125661.0 218819.0 7173.0 2531.0 0.0 1187105.0 5413544.0 82.5 1.8 3.2 0.1 0.0 0.0 17.4 79.3 100 100 100.00 38 682917200 27.1 22.5 21.2 29.2 0.0 35.7 18.2 smartseq 500916 SRR2049496 SRP059035 SRS951875 SRX1047576 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702744: LC-MBT-15_SC20; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702744 GSM1702744 LC-MBT-15_SC20 1365263800 6826319 2015-06-05 16:18:03 898728674 1365263800 6826319 2 6826319 index:0,count:6826319,average:100,stdev:0|index:1,count:6826319,average:100,stdev:0 GSM1702744_r1 2.77 4.24 0.02 1078228426 1410891938 1037974943 1363390963 130.85 131.35 6158461 5299169 255.706 1386.119 164 26695 76.18 79.32 6573864 4691349 6573864 4691349 74.68 74.97 6573864 4599068 6573864 4434008 117092211 10.86 1.45 0 3.57 0 0.09 0 0.02 0 0.00 0 9.67 0 6158461 0 200 0 196.42 0 1.57 0 0.01 0 1.48 0 0.01 0 146.28 0 0.39 0 99277 0 6826319 0 243770 0 6248 0 1567 0 0 0 660043 0 1014 0 0 0 16834 0 2198184 0 16190 0 2232222 0 86.65 0 5914691 0 51312 2160914 42.113228874337 6826319.0 6158461.0 99277.0 243770.0 6248.0 1567.0 0.0 660043.0 5914691.0 90.2 1.5 3.6 0.1 0.0 0.0 9.7 86.6 100 100 100.00 38 682631900 27.4 21.9 21.6 29.1 0.0 35.8 18.4 smartseq 500926 SRR2049497 SRP059035 SRS951874 SRX1047577 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702745: LC-MBT-15_SC22; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702745 GSM1702745 LC-MBT-15_SC22 1396292400 6981462 2015-06-05 16:18:03 930419255 1396292400 6981462 2 6981462 index:0,count:6981462,average:100,stdev:0|index:1,count:6981462,average:100,stdev:0 GSM1702745_r1 2.23 4.23 0.02 1070445212 1387407299 1033239925 1342858273 129.61 129.97 6310696 5472085 240.164 1343.032 153 28658 75.67 78.57 6698408 4775279 6698408 4775279 73.95 74.18 6698408 4666477 6698408 4508526 118057280 11.03 1.41 0 3.34 0 0.09 0 0.04 0 0.00 0 9.48 0 6310696 0 200 0 196.15 0 1.57 0 0.01 0 1.50 0 0.01 0 151.41 0 0.41 0 98236 0 6981462 0 232963 0 6525 0 2532 0 0 0 661709 0 1136 0 0 0 15667 0 2294569 0 15858 0 2327230 0 87.06 0 6077733 0 57314 2186818 38.155040653244 6981462.0 6310696.0 98236.0 232963.0 6525.0 2532.0 0.0 661709.0 6077733.0 90.4 1.4 3.3 0.1 0.0 0.0 9.5 87.1 100 100 100.00 38 698146200 27.5 21.9 21.6 29.0 0.0 35.9 18.9 smartseq 500933 SRR2049498 SRP059035 SRS951873 SRX1047578 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702746: LC-MBT-15_SC24; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702746 GSM1702746 LC-MBT-15_SC24 1206708600 6033543 2015-06-05 16:18:03 799713468 1206708600 6033543 2 6033543 index:0,count:6033543,average:100,stdev:0|index:1,count:6033543,average:100,stdev:0 GSM1702746_r1 2.12 3.96 0.01 919044066 1123360874 890697567 1090988044 122.23 122.49 5405333 4856994 246.619 1242.325 143 24644 59.21 61.25 5717012 3200700 5717012 3200700 58.11 58.12 5717012 3140931 5717012 3036783 207290767 22.56 1.68 0 2.98 0 0.14 0 0.05 0 0.00 0 10.22 0 5405333 0 200 0 196.29 0 1.58 0 0.02 0 1.48 0 0.01 0 143.85 0 0.41 0 101293 0 6033543 0 180025 0 8261 0 3270 0 0 0 616679 0 900 0 0 0 11793 0 1426495 0 13940 0 1453128 0 86.60 0 5225308 0 49998 1363708 27.275251010040 6033543.0 5405333.0 101293.0 180025.0 8261.0 3270.0 0.0 616679.0 5225308.0 89.6 1.7 3.0 0.1 0.1 0.0 10.2 86.6 100 100 100.00 38 603354300 28.0 21.4 21.0 29.7 0.0 35.8 18.4 smartseq 500941 SRR2049499 SRP059035 SRS951872 SRX1047579 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702747: LC-MBT-15_SC25; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702747 GSM1702747 LC-MBT-15_SC25 713097600 3565488 2015-06-05 16:18:03 476360749 713097600 3565488 2 3565488 index:0,count:3565488,average:100,stdev:0|index:1,count:3565488,average:100,stdev:0 GSM1702747_r1 2.97 3.89 0.03 504184113 625481209 484971106 604306852 124.06 124.61 3048885 2763091 229.861 1863.604 130 15308 58.41 60.98 3272290 1780768 3272290 1780768 57.33 57.7 3272290 1747989 3272290 1684866 110212132 21.86 2.77 0 3.61 0 0.14 0 0.04 0 0.00 0 14.31 0 3048885 0 200 0 194.87 0 1.59 0 0.02 0 1.49 0 0.01 0 124.62 0 0.65 0 98894 0 3565488 0 128632 0 4868 0 1419 0 0 0 510316 0 369 0 0 0 7550 0 739214 0 8704 0 755837 0 81.90 0 2920253 0 60951 698291 11.456596282260 3565488.0 3048885.0 98894.0 128632.0 4868.0 1419.0 0.0 510316.0 2920253.0 85.5 2.8 3.6 0.1 0.0 0.0 14.3 81.9 100 100 100.00 38 356548800 27.0 22.1 21.4 29.5 0.0 35.4 17.5 smartseq 501764 SRR2049500 SRP059035 SRS951871 SRX1047580 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702748: LC-MBT-15_SC34; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702748 GSM1702748 LC-MBT-15_SC34 1716815200 8584076 2015-06-05 16:18:03 1138672245 1716815200 8584076 2 8584076 index:0,count:8584076,average:100,stdev:0|index:1,count:8584076,average:100,stdev:0 GSM1702748_r1 2.45 4.29 0.02 1380055609 1729494167 1335920506 1677963526 125.32 125.6 7839369 6837190 255.989 1333.642 174 33812 65.92 68.21 8263011 5167731 8263011 5167731 64.89 64.79 8263011 5086852 8263011 4908815 246604706 17.87 1.29 0 3.06 0 0.07 0 0.04 0 0.00 0 8.56 0 7839369 0 200 0 196.79 0 1.59 0 0.02 0 1.50 0 0.01 0 170.73 0 0.38 0 110824 0 8584076 0 263091 0 6037 0 3832 0 0 0 734838 0 991 0 0 0 18396 0 2505817 0 18999 0 2544203 0 88.26 0 7576278 0 48336 2461144 50.917411453161 8584076.0 7839369.0 110824.0 263091.0 6037.0 3832.0 0.0 734838.0 7576278.0 91.3 1.3 3.1 0.1 0.0 0.0 8.6 88.3 100 100 100.00 38 858407600 27.7 21.8 21.5 29.0 0.0 35.9 19.0 smartseq 501772 SRR2049501 SRP059035 SRS951870 SRX1047581 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702749: LC-MBT-15_SC41; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702749 GSM1702749 LC-MBT-15_SC41 1548580200 7742901 2015-06-05 16:18:03 1036854624 1548580200 7742901 2 7742901 index:0,count:7742901,average:100,stdev:0|index:1,count:7742901,average:100,stdev:0 GSM1702749_r1 2.88 4.04 0.01 1243495674 1604627871 1204525353 1557616122 129.04 129.31 7063590 6128733 255.443 1347.628 175 30683 71.37 73.8 7449647 5041481 7449647 5041481 69.95 69.97 7449647 4941011 7449647 4779987 177829638 14.30 1.28 0 3.00 0 0.06 0 0.04 0 0.00 0 8.68 0 7063590 0 200 0 196.70 0 1.57 0 0.02 0 1.44 0 0.01 0 196.30 0 0.42 0 99045 0 7742901 0 232279 0 4791 0 2817 0 0 0 671703 0 1260 0 0 0 17578 0 2366069 0 17186 0 2402093 0 88.23 0 6831311 0 48707 2318893 47.609029502946 7742901.0 7063590.0 99045.0 232279.0 4791.0 2817.0 0.0 671703.0 6831311.0 91.2 1.3 3.0 0.1 0.0 0.0 8.7 88.2 100 100 100.00 38 774290100 27.6 21.9 21.7 28.8 0.0 35.9 19.2 smartseq 501797 SRR2049504 SRP059035 SRS951867 SRX1047584 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702752: LC-MBT-15_SC50; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702752 GSM1702752 LC-MBT-15_SC50 1352569200 6762846 2015-06-05 16:18:03 899124396 1352569200 6762846 2 6762846 index:0,count:6762846,average:100,stdev:0|index:1,count:6762846,average:100,stdev:0 GSM1702752_r1 2.5 3.92 0.02 874140660 1102080293 841538315 1065312582 126.08 126.59 5498618 5002318 219.327 1259.997 111 28792 62.48 65.16 5930716 3435617 5930716 3435617 61.05 61.41 5930716 3356781 5930716 3237694 179111285 20.49 1.86 0 3.35 0 0.14 0 0.03 0 0.00 0 18.52 0 5498618 0 200 0 193.98 0 1.55 0 0.02 0 1.48 0 0.01 0 114.30 0 0.45 0 125976 0 6762846 0 226280 0 9605 0 2165 0 0 0 1252458 0 630 0 0 0 11163 0 1429170 0 12907 0 1453870 0 77.96 0 5272338 0 51749 1311457 25.342653964328 6762846.0 5498618.0 125976.0 226280.0 9605.0 2165.0 0.0 1252458.0 5272338.0 81.3 1.9 3.3 0.1 0.0 0.0 18.5 78.0 100 100 100.00 38 676284600 27.1 22.4 21.1 29.5 0.0 35.5 17.6 smartseq 501805 SRR2049505 SRP059035 SRS951866 SRX1047585 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702753: LC-MBT-15_SC51; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702753 GSM1702753 LC-MBT-15_SC51 731195200 3655976 2015-06-05 16:18:03 484838476 731195200 3655976 2 3655976 index:0,count:3655976,average:100,stdev:0|index:1,count:3655976,average:100,stdev:0 GSM1702753_r1 2.91 3.89 0.03 469313668 589855409 450867581 569534706 125.68 126.32 2950401 2702281 220.768 1679.129 103 15464 59.92 62.71 3190737 1767971 3190737 1767971 58.39 58.86 3190737 1722727 3190737 1659378 95295434 20.31 2.66 0 3.59 0 0.16 0 0.04 0 0.00 0 19.10 0 2950401 0 200 0 193.70 0 1.56 0 0.02 0 1.50 0 0.01 0 98.96 0 0.54 0 97131 0 3655976 0 131297 0 5706 0 1460 0 0 0 698409 0 343 0 0 0 6579 0 680172 0 7462 0 694556 0 77.11 0 2819104 0 50764 626844 12.348199511465 3655976.0 2950401.0 97131.0 131297.0 5706.0 1460.0 0.0 698409.0 2819104.0 80.7 2.7 3.6 0.2 0.0 0.0 19.1 77.1 100 100 100.00 38 365597600 26.8 22.6 21.3 29.4 0.0 35.3 17.4 smartseq 501813 SRR2049506 SRP059035 SRS951865 SRX1047586 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702754: LC-MBT-15_SC52; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702754 GSM1702754 LC-MBT-15_SC52 1547626200 7738131 2015-06-05 16:18:03 1020750852 1547626200 7738131 2 7738131 index:0,count:7738131,average:100,stdev:0|index:1,count:7738131,average:100,stdev:0 GSM1702754_r1 4.74 4.41 0.02 1207713696 1608092708 1168413908 1560756391 133.15 133.58 6840738 5922234 263.373 1295.812 173 28965 75.16 77.85 7269115 5141826 7269115 5141826 72.91 73.17 7269115 4987730 7269115 4832455 146623310 12.14 1.26 0 3.05 0 0.07 0 0.01 0 0.00 0 11.51 0 6840738 0 200 0 196.69 0 1.54 0 0.02 0 1.49 0 0.01 0 153.91 0 0.39 0 97466 0 7738131 0 236252 0 5681 0 1071 0 0 0 890641 0 1367 0 0 0 18054 0 2361986 0 16905 0 2398312 0 85.35 0 6604486 0 39401 2316827 58.801223319205 7738131.0 6840738.0 97466.0 236252.0 5681.0 1071.0 0.0 890641.0 6604486.0 88.4 1.3 3.1 0.1 0.0 0.0 11.5 85.3 100 100 100.00 38 773813100 27.0 22.3 22.0 28.7 0.0 35.7 18.6 smartseq 501821 SRR2049507 SRP059035 SRS951864 SRX1047587 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702755: LC-MBT-15_SC54; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702755 GSM1702755 LC-MBT-15_SC54 1834468000 9172340 2015-06-05 16:18:03 1213397669 1834468000 9172340 2 9172340 index:0,count:9172340,average:100,stdev:0|index:1,count:9172340,average:100,stdev:0 GSM1702755_r1 2.31 4.27 0.02 1232152059 1498590306 1191242748 1452443802 121.62 121.93 7648745 6951300 224.551 1131.101 110 38415 57.54 59.68 8142467 4401011 8142467 4401011 56.52 56.47 8142467 4323384 8142467 4164195 273392686 22.19 1.46 0 2.99 0 0.08 0 0.04 0 0.00 0 16.49 0 7648745 0 200 0 194.72 0 1.60 0 0.02 0 1.49 0 0.01 0 154.30 0 0.39 0 134371 0 9172340 0 274629 0 7098 0 3914 0 0 0 1512583 0 817 0 0 0 15019 0 1948448 0 18151 0 1982435 0 80.40 0 7374116 0 46679 1801706 38.597784871141 9172340.0 7648745.0 134371.0 274629.0 7098.0 3914.0 0.0 1512583.0 7374116.0 83.4 1.5 3.0 0.1 0.0 0.0 16.5 80.4 100 100 100.00 38 917234000 27.7 22.2 21.1 29.1 0.0 35.8 18.5 smartseq 501830 SRR2049508 SRP059035 SRS951863 SRX1047588 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702756: LC-MBT-15_SC55; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702756 GSM1702756 LC-MBT-15_SC55 1012066200 5060331 2015-06-05 16:18:03 669376267 1012066200 5060331 2 5060331 index:0,count:5060331,average:100,stdev:0|index:1,count:5060331,average:100,stdev:0 GSM1702756_r1 2.5 4.08 0.02 774530858 964832442 748086160 935138294 124.57 125.0 4466350 3981653 250.289 1686.823 164 19856 62.79 65.19 4753676 2804273 4753676 2804273 61.52 61.72 4753676 2747727 4753676 2655337 140941127 18.20 2.25 0 3.25 0 0.11 0 0.05 0 0.00 0 11.58 0 4466350 0 200 0 195.99 0 1.58 0 0.02 0 1.48 0 0.01 0 157.04 0 0.50 0 114017 0 5060331 0 164419 0 5418 0 2342 0 0 0 586221 0 523 0 0 0 10183 0 1186141 0 12120 0 1208967 0 85.01 0 4301931 0 57005 1156952 20.295623190948 5060331.0 4466350.0 114017.0 164419.0 5418.0 2342.0 0.0 586221.0 4301931.0 88.3 2.3 3.2 0.1 0.0 0.0 11.6 85.0 100 100 100.00 38 506033100 27.3 22.0 21.5 29.2 0.0 35.6 17.9 smartseq 501837 SRR2049509 SRP059035 SRS951862 SRX1047589 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702757: LC-MBT-15_SC56; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702757 GSM1702757 LC-MBT-15_SC56 772917800 3864589 2015-06-05 16:18:03 510646910 772917800 3864589 2 3864589 index:0,count:3864589,average:100,stdev:0|index:1,count:3864589,average:100,stdev:0 GSM1702757_r1 3.23 3.8 0.03 544759752 681581782 523797595 658708887 125.12 125.76 3202145 2936023 245.906 2181.777 160 15472 55.17 57.64 3455890 1766726 3455890 1766726 54.31 54.69 3455890 1739067 3455890 1676150 135961244 24.96 4.11 0 3.55 0 0.16 0 0.04 0 0.00 0 16.95 0 3202145 0 200 0 194.69 0 1.56 0 0.02 0 1.49 0 0.01 0 106.20 0 0.70 0 158869 0 3864589 0 137071 0 6069 0 1432 0 0 0 654943 0 209 0 0 0 6877 0 597970 0 9286 0 614342 0 79.31 0 3065074 0 56821 584109 10.279808521497 3864589.0 3202145.0 158869.0 137071.0 6069.0 1432.0 0.0 654943.0 3065074.0 82.9 4.1 3.5 0.2 0.0 0.0 16.9 79.3 100 100 100.00 38 386458900 26.7 22.1 21.3 29.8 0.0 35.1 16.6 smartseq 501892 SRR2049510 SRP059035 SRS951861 SRX1047590 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702758: LC-MBT-15_SC57; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702758 GSM1702758 LC-MBT-15_SC57 821314400 4106572 2015-06-05 16:18:03 549610104 821314400 4106572 2 4106572 index:0,count:4106572,average:100,stdev:0|index:1,count:4106572,average:100,stdev:0 GSM1702758_r1 2.02 3.98 0.03 522427501 670397879 501286080 646004920 128.32 128.87 3314541 2978240 215.865 1545.221 106 17707 67.57 70.76 3573643 2239624 3573643 2239624 66.13 66.63 3573643 2191824 3573643 2108922 78193261 14.97 1.92 0 3.63 0 0.13 0 0.03 0 0.00 0 19.13 0 3314541 0 200 0 193.61 0 1.55 0 0.01 0 1.49 0 0.01 0 114.60 0 0.55 0 78814 0 4106572 0 149258 0 5335 0 1111 0 0 0 785585 0 350 0 0 0 8075 0 966200 0 8721 0 983346 0 77.08 0 3165283 0 55806 884908 15.856861269398 4106572.0 3314541.0 78814.0 149258.0 5335.0 1111.0 0.0 785585.0 3165283.0 80.7 1.9 3.6 0.1 0.0 0.0 19.1 77.1 100 100 100.00 38 410657200 27.0 22.7 21.5 28.9 0.0 35.5 17.9 smartseq 501901 SRR2049511 SRP059035 SRS951860 SRX1047591 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702759: LC-MBT-15_SC58; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702759 GSM1702759 LC-MBT-15_SC58 1213552200 6067761 2015-06-05 16:18:03 797870183 1213552200 6067761 2 6067761 index:0,count:6067761,average:100,stdev:0|index:1,count:6067761,average:100,stdev:0 GSM1702759_r1 1.65 4.04 0.02 803258154 996883114 775158923 965534833 124.1 124.56 4997413 4509258 227.066 1432.426 101 24804 59.91 62.34 5338620 2993934 5338620 2993934 58.53 58.77 5338620 2924885 5338620 2822321 180161777 22.43 1.87 0 3.21 0 0.12 0 0.04 0 0.00 0 17.49 0 4997413 0 200 0 194.26 0 1.58 0 0.02 0 1.50 0 0.01 0 130.02 0 0.47 0 113235 0 6067761 0 195006 0 7015 0 2158 0 0 0 1061175 0 514 0 0 0 11216 0 1326109 0 12737 0 1350576 0 79.15 0 4802407 0 54935 1222756 22.258232456540 6067761.0 4997413.0 113235.0 195006.0 7015.0 2158.0 0.0 1061175.0 4802407.0 82.4 1.9 3.2 0.1 0.0 0.0 17.5 79.1 100 100 100.00 38 606776100 27.1 22.5 21.2 29.2 0.0 35.6 18.0 smartseq 501909 SRR2049512 SRP059035 SRS951859 SRX1047592 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702760: LC-MBT-15_SC59; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702760 GSM1702760 LC-MBT-15_SC59 869048400 4345242 2015-06-05 16:18:03 576805797 869048400 4345242 2 4345242 index:0,count:4345242,average:100,stdev:0|index:1,count:4345242,average:100,stdev:0 GSM1702760_r1 2.43 4.08 0.02 659187716 827224627 636120391 801624977 125.49 126.02 3804630 3402809 254.936 1554.972 160 16862 63.49 66.02 4064174 2415505 4064174 2415505 62.38 62.69 4064174 2373495 4064174 2293938 120300722 18.25 2.25 0 3.35 0 0.17 0 0.03 0 0.00 0 12.24 0 3804630 0 200 0 196.01 0 1.58 0 0.02 0 1.52 0 0.01 0 138.43 0 0.50 0 97807 0 4345242 0 145725 0 7247 0 1329 0 0 0 532036 0 459 0 0 0 9016 0 995622 0 9738 0 1014835 0 84.20 0 3658905 0 53878 970226 18.007832510487 4345242.0 3804630.0 97807.0 145725.0 7247.0 1329.0 0.0 532036.0 3658905.0 87.6 2.3 3.4 0.2 0.0 0.0 12.2 84.2 100 100 100.00 38 434524200 27.5 21.6 21.1 29.8 0.0 35.6 17.8 smartseq 501916 SRR2049513 SRP059035 SRS951858 SRX1047593 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702761: LC-MBT-15_SC60; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702761 GSM1702761 LC-MBT-15_SC60 1486466200 7432331 2015-06-05 16:18:03 982123763 1486466200 7432331 2 7432331 index:0,count:7432331,average:100,stdev:0|index:1,count:7432331,average:100,stdev:0 GSM1702761_r1 1.92 3.92 0.02 1144458952 1444808783 1108633003 1402670359 126.24 126.52 6712547 5933405 246.223 1356.957 143 29823 65.56 67.84 7097917 4400502 7097917 4400502 64.21 64.26 7097917 4309940 7097917 4168261 203493645 17.78 1.55 0 3.03 0 0.08 0 0.04 0 0.00 0 9.56 0 6712547 0 200 0 196.31 0 1.59 0 0.02 0 1.47 0 0.01 0 182.02 0 0.41 0 115155 0 7432331 0 225496 0 6204 0 2883 0 0 0 710697 0 1440 0 0 0 15467 0 2057638 0 16554 0 2091099 0 87.28 0 6487051 0 56815 1963686 34.562809117311 7432331.0 6712547.0 115155.0 225496.0 6204.0 2883.0 0.0 710697.0 6487051.0 90.3 1.5 3.0 0.1 0.0 0.0 9.6 87.3 100 100 100.00 38 743233100 27.5 21.9 21.5 29.1 0.0 35.8 18.6 smartseq 501925 SRR2049514 SRP059035 SRS951857 SRX1047594 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702762: LC-MBT-15_SC62; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702762 GSM1702762 LC-MBT-15_SC62 1181264200 5906321 2015-06-05 16:18:03 780507624 1181264200 5906321 2 5906321 index:0,count:5906321,average:100,stdev:0|index:1,count:5906321,average:100,stdev:0 GSM1702762_r1 2.65 3.72 0.02 748586017 946744817 718102587 912676683 126.47 127.1 4743274 4339179 217.961 1467.705 106 25777 59.76 62.6 5134368 2834810 5134368 2834810 58.83 59.18 5134368 2790536 5134368 2679936 153606938 20.52 2.17 0 3.63 0 0.14 0 0.03 0 0.00 0 19.52 0 4743274 0 200 0 193.61 0 1.57 0 0.02 0 1.47 0 0.01 0 158.68 0 0.53 0 128385 0 5906321 0 214553 0 8219 0 1627 0 0 0 1153201 0 526 0 0 0 10658 0 1134417 0 12471 0 1158072 0 76.68 0 4528721 0 55901 1046419 18.719146348008 5906321.0 4743274.0 128385.0 214553.0 8219.0 1627.0 0.0 1153201.0 4528721.0 80.3 2.2 3.6 0.1 0.0 0.0 19.5 76.7 100 100 100.00 38 590632100 27.0 22.5 21.3 29.2 0.0 35.5 17.6 smartseq 501932 SRR2049515 SRP059035 SRS951856 SRX1047595 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702763: LC-MBT-15_SC63; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702763 GSM1702763 LC-MBT-15_SC63 1648976600 8244883 2015-06-05 16:18:03 1099767584 1648976600 8244883 2 8244883 index:0,count:8244883,average:100,stdev:0|index:1,count:8244883,average:100,stdev:0 GSM1702763_r1 2.35 3.69 0.02 1317971367 1621691281 1283467693 1583104629 123.04 123.35 7471707 6635541 255.991 1232.968 174 32266 62.92 64.69 7895010 4700844 7895010 4700844 61.63 61.76 7895010 4605081 7895010 4487525 273875218 20.78 1.24 0 2.49 0 0.06 0 0.03 0 0.00 0 9.30 0 7471707 0 200 0 196.95 0 1.67 0 0.02 0 1.49 0 0.01 0 148.41 0 0.41 0 102093 0 8244883 0 205379 0 4548 0 2065 0 0 0 766563 0 1123 0 0 0 16138 0 1989923 0 19275 0 2026459 0 88.13 0 7266328 0 42396 1939774 45.753703179545 8244883.0 7471707.0 102093.0 205379.0 4548.0 2065.0 0.0 766563.0 7266328.0 90.6 1.2 2.5 0.1 0.0 0.0 9.3 88.1 100 100 100.00 38 824488300 28.4 21.1 20.9 29.6 0.0 36.0 19.0 smartseq 501940 SRR2049516 SRP059035 SRS951855 SRX1047596 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702764: LC-MBT-15_SC64; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702764 GSM1702764 LC-MBT-15_SC64 1556408200 7782041 2015-06-05 16:18:03 1023880959 1556408200 7782041 2 7782041 index:0,count:7782041,average:100,stdev:0|index:1,count:7782041,average:100,stdev:0 GSM1702764_r1 2.77 4.08 0.02 1192634747 1506636033 1153750317 1461188773 126.33 126.65 7016628 6237199 245.228 1204.303 152 32020 65.98 68.36 7427775 4629276 7427775 4629276 64.63 64.62 7427775 4534804 7427775 4375475 203974103 17.10 1.43 0 3.15 0 0.09 0 0.04 0 0.00 0 9.70 0 7016628 0 200 0 196.37 0 1.57 0 0.02 0 1.47 0 0.01 0 155.64 0 0.38 0 111106 0 7782041 0 245192 0 7150 0 3115 0 0 0 755148 0 876 0 0 0 14409 0 2124923 0 16562 0 2156770 0 87.01 0 6771436 0 51095 2030416 39.738056561307 7782041.0 7016628.0 111106.0 245192.0 7150.0 3115.0 0.0 755148.0 6771436.0 90.2 1.4 3.2 0.1 0.0 0.0 9.7 87.0 100 100 100.00 38 778204100 27.6 21.7 21.4 29.3 0.0 35.9 18.7 smartseq 501949 SRR2049517 SRP059035 SRS951854 SRX1047597 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702765: LC-MBT-15_SC65; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702765 GSM1702765 LC-MBT-15_SC65 540318200 2701591 2015-06-05 16:18:03 358774778 540318200 2701591 2 2701591 index:0,count:2701591,average:100,stdev:0|index:1,count:2701591,average:100,stdev:0 GSM1702765_r1 3.23 3.94 0.02 393033391 492532206 377942172 475828687 125.32 125.9 2289117 2056620 248.066 1932.471 160 10843 60.84 63.53 2463279 1392711 2463279 1392711 59.86 60.26 2463279 1370314 2463279 1320976 78412090 19.95 3.38 0 3.59 0 0.17 0 0.04 0 0.00 0 15.06 0 2289117 0 200 0 195.14 0 1.55 0 0.02 0 1.50 0 0.01 0 103.47 0 0.60 0 91430 0 2701591 0 96982 0 4620 0 1053 0 0 0 406801 0 288 0 0 0 5043 0 562744 0 6382 0 574457 0 81.14 0 2192135 0 52761 548659 10.398949981994 2701591.0 2289117.0 91430.0 96982.0 4620.0 1053.0 0.0 406801.0 2192135.0 84.7 3.4 3.6 0.2 0.0 0.0 15.1 81.1 100 100 100.00 38 270159100 26.8 22.0 21.3 29.8 0.0 35.3 17.0 smartseq 501957 SRR2049518 SRP059035 SRS951853 SRX1047598 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702766: LC-MBT-15_SC67; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702766 GSM1702766 LC-MBT-15_SC67 1450794600 7253973 2015-06-05 16:18:03 953016543 1450794600 7253973 2 7253973 index:0,count:7253973,average:100,stdev:0|index:1,count:7253973,average:100,stdev:0 GSM1702766_r1 2.97 3.88 0.02 1101585866 1438972371 1061581011 1392007605 130.63 131.13 6494888 5659038 244.521 1308.948 153 29254 76.56 79.68 6939545 4972291 6939545 4972291 74.96 75.37 6939545 4868596 6939545 4702985 116914314 10.61 1.53 0 3.51 0 0.14 0 0.04 0 0.00 0 10.28 0 6494888 0 200 0 196.10 0 1.57 0 0.02 0 1.49 0 0.01 0 148.38 0 0.38 0 111282 0 7253973 0 254896 0 9945 0 3147 0 0 0 745993 0 864 0 0 0 15968 0 2259163 0 15130 0 2291125 0 86.02 0 6239992 0 52573 2153530 40.962661442185 7253973.0 6494888.0 111282.0 254896.0 9945.0 3147.0 0.0 745993.0 6239992.0 89.5 1.5 3.5 0.1 0.0 0.0 10.3 86.0 100 100 100.00 38 725397300 27.6 21.6 21.3 29.6 0.0 35.8 18.3 smartseq 501965 SRR2049519 SRP059035 SRS951852 SRX1047599 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702767: LC-MBT-15_SC71; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702767 GSM1702767 LC-MBT-15_SC71 1718485200 8592426 2015-06-05 16:18:03 1130071257 1718485200 8592426 2 8592426 index:0,count:8592426,average:100,stdev:0|index:1,count:8592426,average:100,stdev:0 GSM1702767_r1 3.36 4.39 0.01 1377101877 1725631541 1337249273 1679161855 125.31 125.57 7790519 6832346 262.081 1294.158 175 32778 66.3 68.39 8188254 5164859 8188254 5164859 64.92 64.94 8188254 5057581 8188254 4904005 240693688 17.48 1.31 0 2.78 0 0.09 0 0.04 0 0.00 0 9.21 0 7790519 0 200 0 196.86 0 1.56 0 0.02 0 1.48 0 0.01 0 157.82 0 0.37 0 112527 0 8592426 0 238532 0 7664 0 3202 0 0 0 791041 0 1204 0 0 0 17047 0 2333728 0 18851 0 2370830 0 87.89 0 7551987 0 41093 2284662 55.597352347115 8592426.0 7790519.0 112527.0 238532.0 7664.0 3202.0 0.0 791041.0 7551987.0 90.7 1.3 2.8 0.1 0.0 0.0 9.2 87.9 100 100 100.00 38 859242600 28.0 21.3 21.0 29.6 0.0 35.9 18.6 smartseq 502021 SRR2049520 SRP059035 SRS951851 SRX1047600 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702768: LC-MBT-15_SC72; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702768 GSM1702768 LC-MBT-15_SC72 1773267400 8866337 2015-06-05 16:18:03 1171772636 1773267400 8866337 2 8866337 index:0,count:8866337,average:100,stdev:0|index:1,count:8866337,average:100,stdev:0 GSM1702768_r1 3.82 3.81 0.01 1417577085 1910728919 1362941581 1844489078 134.79 135.33 8068219 6752895 259.424 1457.554 175 34587 84.19 87.76 8597572 6792942 8597572 6792942 82.65 83.08 8597572 6668038 8597572 6431148 81319748 5.74 1.28 0 3.70 0 0.08 0 0.02 0 0.00 0 8.90 0 8068219 0 200 0 196.53 0 1.54 0 0.01 0 1.51 0 0.01 0 155.70 0 0.37 0 113467 0 8866337 0 327736 0 6830 0 1807 0 0 0 789481 0 1621 0 0 0 22682 0 3402885 0 19745 0 3446933 0 87.30 0 7740483 0 47472 3326436 70.071536905966 8866337.0 8068219.0 113467.0 327736.0 6830.0 1807.0 0.0 789481.0 7740483.0 91.0 1.3 3.7 0.1 0.0 0.0 8.9 87.3 100 100 100.00 38 886633700 27.2 22.2 22.0 28.6 0.0 35.8 18.9 smartseq 502029 SRR2049521 SRP059035 SRS951850 SRX1047601 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702769: LC-MBT-15_SC73; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702769 GSM1702769 LC-MBT-15_SC73 1542876200 7714381 2015-06-05 16:18:03 1020647526 1542876200 7714381 2 7714381 index:0,count:7714381,average:100,stdev:0|index:1,count:7714381,average:100,stdev:0 GSM1702769_r1 2.7 4.47 0.02 1040698005 1312748975 1005827793 1271748328 126.14 126.44 6451992 5746784 225.711 1273.816 111 31447 67.59 70.17 6859496 4361137 6859496 4361137 66.13 66.28 6859496 4266846 6859496 4119207 154422733 14.84 1.63 0 3.07 0 0.12 0 0.05 0 0.00 0 16.20 0 6451992 0 200 0 194.47 0 1.55 0 0.02 0 1.46 0 0.01 0 143.15 0 0.40 0 126050 0 7714381 0 236786 0 8879 0 3643 0 0 0 1249867 0 782 0 0 0 16044 0 1964621 0 16556 0 1998003 0 80.57 0 6215206 0 51310 1821273 35.495478464237 7714381.0 6451992.0 126050.0 236786.0 8879.0 3643.0 0.0 1249867.0 6215206.0 83.6 1.6 3.1 0.1 0.0 0.0 16.2 80.6 100 100 100.00 38 771438100 27.5 22.1 21.1 29.3 0.0 35.7 18.3 smartseq 502036 SRR2049522 SRP059035 SRS951849 SRX1047602 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702770: LC-MBT-15_SC74; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702770 GSM1702770 LC-MBT-15_SC74 1635410600 8177053 2015-06-05 16:18:03 1078973330 1635410600 8177053 2 8177053 index:0,count:8177053,average:100,stdev:0|index:1,count:8177053,average:100,stdev:0 GSM1702770_r1 3.16 3.51 0.02 1254298211 1603922168 1212240822 1554093110 127.87 128.2 7386781 6492809 243.919 1299.185 143 33771 68.32 70.85 7822706 5046287 7822706 5046287 66.84 66.93 7822706 4937256 7822706 4767054 203092705 16.19 1.53 0 3.24 0 0.11 0 0.04 0 0.00 0 9.52 0 7386781 0 200 0 196.24 0 1.56 0 0.02 0 1.51 0 0.01 0 156.58 0 0.39 0 125014 0 8177053 0 264570 0 9277 0 2933 0 0 0 778062 0 807 0 0 0 17610 0 2394972 0 18835 0 2432224 0 87.10 0 7122211 0 55388 2289020 41.327002238752 8177053.0 7386781.0 125014.0 264570.0 9277.0 2933.0 0.0 778062.0 7122211.0 90.3 1.5 3.2 0.1 0.0 0.0 9.5 87.1 100 100 100.00 38 817705300 27.5 21.9 21.5 29.0 0.0 35.9 18.8 smartseq 502044 SRR2049523 SRP059035 SRS951848 SRX1047603 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702771: LC-MBT-15_SC75; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702771 GSM1702771 LC-MBT-15_SC75 1545135600 7725678 2015-06-05 16:18:03 1032046889 1545135600 7725678 2 7725678 index:0,count:7725678,average:100,stdev:0|index:1,count:7725678,average:100,stdev:0 GSM1702771_r1 4.22 3.71 0.02 1036099871 1310229555 1002402774 1270900674 126.46 126.79 6454849 5876655 219.476 1090.780 111 32983 61.57 63.82 6848434 3974284 6848434 3974284 60.19 60.2 6848434 3885440 6848434 3749314 226748238 21.88 1.61 0 2.94 0 0.13 0 0.04 0 0.00 0 16.28 0 6454849 0 200 0 194.50 0 1.54 0 0.02 0 1.47 0 0.01 0 145.61 0 0.42 0 124752 0 7725678 0 227040 0 9672 0 3316 0 0 0 1257841 0 670 0 0 0 11785 0 1650243 0 15831 0 1678529 0 80.61 0 6227809 0 43899 1528091 34.809243946331 7725678.0 6454849.0 124752.0 227040.0 9672.0 3316.0 0.0 1257841.0 6227809.0 83.6 1.6 2.9 0.1 0.0 0.0 16.3 80.6 100 100 100.00 38 772567800 27.5 22.2 21.0 29.3 0.0 35.8 18.6 smartseq 502052 SRR2049524 SRP059035 SRS951847 SRX1047604 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702772: LC-MBT-15_SC76; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702772 GSM1702772 LC-MBT-15_SC76 2017539400 10087697 2015-06-05 16:18:03 1322976165 2017539400 10087697 2 10087697 index:0,count:10087697,average:100,stdev:0|index:1,count:10087697,average:100,stdev:0 GSM1702772_r1 2.26 4.01 0.02 1563999984 1930705995 1517953573 1876543432 123.45 123.62 9150645 8135481 243.779 1175.701 143 41073 62.73 64.75 9596446 5740531 9596446 5740531 61.51 61.35 9596446 5628618 9596446 5439482 298889880 19.11 1.41 0 2.82 0 0.07 0 0.03 0 0.00 0 9.18 0 9150645 0 200 0 196.56 0 1.57 0 0.02 0 1.48 0 0.01 0 178.02 0 0.36 0 141743 0 10087697 0 284639 0 7313 0 3515 0 0 0 926224 0 1259 0 0 0 18748 0 2713257 0 21560 0 2754824 0 87.89 0 8866006 0 44477 2604341 58.554781122828 10087697.0 9150645.0 141743.0 284639.0 7313.0 3515.0 0.0 926224.0 8866006.0 90.7 1.4 2.8 0.1 0.0 0.0 9.2 87.9 100 100 100.00 38 1008769700 28.0 21.5 21.1 29.4 0.0 36.0 18.9 smartseq 502060 SRR2049525 SRP059035 SRS951846 SRX1047605 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702773: LC-MBT-15_SC78; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702773 GSM1702773 LC-MBT-15_SC78 1627806000 8139030 2015-06-05 16:18:03 1072071917 1627806000 8139030 2 8139030 index:0,count:8139030,average:100,stdev:0|index:1,count:8139030,average:100,stdev:0 GSM1702773_r1 3.98 3.91 0.02 1256114845 1698223923 1208302437 1641428371 135.2 135.85 7373410 6140839 244.283 1570.617 152 32453 85.03 88.61 7851457 6269335 7851457 6269335 83.22 83.79 7851457 6136399 7851457 5927973 71240674 5.67 1.43 0 3.66 0 0.10 0 0.05 0 0.00 0 9.25 0 7373410 0 200 0 196.04 0 1.56 0 0.01 0 1.47 0 0.01 0 187.82 0 0.39 0 116485 0 8139030 0 298221 0 8244 0 4158 0 0 0 753218 0 1386 0 0 0 25298 0 3319415 0 23868 0 3369967 0 86.93 0 7075189 0 62793 3169020 50.467727294444 8139030.0 7373410.0 116485.0 298221.0 8244.0 4158.0 0.0 753218.0 7075189.0 90.6 1.4 3.7 0.1 0.1 0.0 9.3 86.9 100 100 100.00 38 813903000 27.1 22.3 22.0 28.6 0.0 35.8 18.9 smartseq 502068 SRR2049526 SRP059035 SRS951845 SRX1047606 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702774: LC-MBT-15_SC79; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702774 GSM1702774 LC-MBT-15_SC79 1649573200 8247866 2015-06-05 16:18:03 1089099362 1649573200 8247866 2 8247866 index:0,count:8247866,average:100,stdev:0|index:1,count:8247866,average:100,stdev:0 GSM1702774_r1 2.46 4.14 0.01 1323055541 1677354605 1279665529 1625423570 126.78 127.02 7533969 6548266 255.103 1333.068 163 32875 68.26 70.69 7954952 5142748 7954952 5142748 66.93 66.9 7954952 5042114 7954952 4866847 212372341 16.05 1.38 0 3.14 0 0.07 0 0.03 0 0.00 0 8.55 0 7533969 0 200 0 196.69 0 1.56 0 0.02 0 1.45 0 0.01 0 151.49 0 0.38 0 113470 0 8247866 0 259328 0 5847 0 2547 0 0 0 705503 0 1410 0 0 0 16952 0 2486485 0 19494 0 2524341 0 88.20 0 7274641 0 50636 2435570 48.099573426021 8247866.0 7533969.0 113470.0 259328.0 5847.0 2547.0 0.0 705503.0 7274641.0 91.3 1.4 3.1 0.1 0.0 0.0 8.6 88.2 100 100 100.00 38 824786600 27.7 21.8 21.5 28.9 0.0 35.9 19.0 smartseq 502076 SRR2049527 SRP059035 SRS951844 SRX1047607 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702775: LC-MBT-15_SC80; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702775 GSM1702775 LC-MBT-15_SC80 1671626200 8358131 2015-06-05 16:18:03 1105553551 1671626200 8358131 2 8358131 index:0,count:8358131,average:100,stdev:0|index:1,count:8358131,average:100,stdev:0 GSM1702775_r1 3.82 4.24 0.01 1311005211 1692641675 1270166572 1645825261 129.11 129.58 7498161 6661781 253.646 1231.284 164 32919 68.77 71.12 7962744 5156196 7962744 5156196 67.37 67.58 7962744 5051449 7962744 4900117 221716340 16.91 1.54 0 2.96 0 0.10 0 0.04 0 0.00 0 10.15 0 7498161 0 200 0 196.49 0 1.60 0 0.02 0 1.48 0 0.01 0 146.06 0 0.39 0 128323 0 8358131 0 247725 0 8206 0 3666 0 0 0 848098 0 1240 0 0 0 15335 0 2146204 0 19175 0 2181954 0 86.75 0 7250436 0 42033 2104339 50.063973544596 8358131.0 7498161.0 128323.0 247725.0 8206.0 3666.0 0.0 848098.0 7250436.0 89.7 1.5 3.0 0.1 0.0 0.0 10.1 86.7 100 100 100.00 38 835813100 27.7 21.5 21.3 29.5 0.0 35.7 18.1 smartseq 502084 SRR2049528 SRP059035 SRS951843 SRX1047608 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702776: LC-MBT-15_SC84; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702776 GSM1702776 LC-MBT-15_SC84 1363102000 6815510 2015-06-05 16:18:03 902422898 1363102000 6815510 2 6815510 index:0,count:6815510,average:100,stdev:0|index:1,count:6815510,average:100,stdev:0 GSM1702776_r1 3.26 4.29 0.02 1038880751 1328447392 999542741 1282248074 127.87 128.28 6117995 5355907 244.130 1358.141 133 27448 71.48 74.5 6546709 4372875 6546709 4372875 70.21 70.35 6546709 4295560 6546709 4129313 135411285 13.03 1.58 0 3.64 0 0.12 0 0.04 0 0.00 0 10.07 0 6117995 0 200 0 196.12 0 1.57 0 0.02 0 1.48 0 0.01 0 129.14 0 0.40 0 107743 0 6815510 0 248345 0 8294 0 2598 0 0 0 686623 0 799 0 0 0 14934 0 2051900 0 15052 0 2082685 0 86.12 0 5869650 0 55213 1966007 35.607682973213 6815510.0 6117995.0 107743.0 248345.0 8294.0 2598.0 0.0 686623.0 5869650.0 89.8 1.6 3.6 0.1 0.0 0.0 10.1 86.1 100 100 100.00 38 681551000 27.5 21.7 21.5 29.3 0.0 35.7 18.4 smartseq 502164 SRR2049532 SRP059035 SRS951839 SRX1047612 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702780: LC-MBT-15_SC92; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702780 GSM1702780 LC-MBT-15_SC92 1748196600 8740983 2015-06-05 16:18:03 1158994818 1748196600 8740983 2 8740983 index:0,count:8740983,average:100,stdev:0|index:1,count:8740983,average:100,stdev:0 GSM1702780_r1 3.04 3.78 0.02 1164708942 1472422805 1124302899 1425426217 126.42 126.78 7258396 6521564 221.949 1216.078 111 36714 64.17 66.69 7744177 4657836 7744177 4657836 62.89 62.86 7744177 4564648 7744177 4390636 212050627 18.21 1.57 0 3.13 0 0.10 0 0.03 0 0.00 0 16.82 0 7258396 0 200 0 194.45 0 1.56 0 0.02 0 1.45 0 0.01 0 145.68 0 0.40 0 136830 0 8740983 0 273623 0 9041 0 2927 0 0 0 1470619 0 827 0 0 0 16594 0 2112554 0 17298 0 2147273 0 79.91 0 6984773 0 46719 1955812 41.863310430446 8740983.0 7258396.0 136830.0 273623.0 9041.0 2927.0 0.0 1470619.0 6984773.0 83.0 1.6 3.1 0.1 0.0 0.0 16.8 79.9 100 100 100.00 38 874098300 27.6 22.1 21.1 29.2 0.0 35.7 18.2 smartseq 502172 SRR2049533 SRP059035 SRS951838 SRX1047613 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702781: LC-MBT-15_SC94; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702781 GSM1702781 LC-MBT-15_SC94 1295935800 6479679 2015-06-05 16:18:03 855242073 1295935800 6479679 2 6479679 index:0,count:6479679,average:100,stdev:0|index:1,count:6479679,average:100,stdev:0 GSM1702781_r1 2.5 4.21 0.02 984072894 1258107720 950241052 1218307832 127.85 128.21 5800836 5111524 244.364 1354.777 143 26488 69.5 72.17 6161066 4031409 6161066 4031409 68.01 68.18 6161066 3945258 6161066 3808560 151501683 15.40 1.66 0 3.32 0 0.11 0 0.03 0 0.00 0 10.34 0 5800836 0 200 0 196.11 0 1.58 0 0.02 0 1.46 0 0.01 0 182.24 0 0.41 0 107545 0 6479679 0 214924 0 6828 0 2234 0 0 0 669781 0 993 0 0 0 13911 0 1850782 0 13706 0 1879392 0 86.21 0 5585912 0 56341 1767975 31.379900960224 6479679.0 5800836.0 107545.0 214924.0 6828.0 2234.0 0.0 669781.0 5585912.0 89.5 1.7 3.3 0.1 0.0 0.0 10.3 86.2 100 100 100.00 38 647967900 27.5 21.8 21.4 29.4 0.0 35.8 18.3 smartseq 502180 SRR2049534 SRP059035 SRS951837 SRX1047614 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702782: LC-MBT-15_SC95; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-MBT-15 GEO Accession;;GSM1702782 GSM1702782 LC-MBT-15_SC95 1413346600 7066733 2015-06-05 16:18:03 937857506 1413346600 7066733 2 7066733 index:0,count:7066733,average:100,stdev:0|index:1,count:7066733,average:100,stdev:0 GSM1702782_r1 2.81 3.77 0.02 1118374908 1387977072 1085746095 1349551833 124.11 124.3 6346865 5639078 261.631 1264.749 175 27117 61.77 63.76 6685218 3920640 6685218 3920640 60.67 60.58 6685218 3850657 6685218 3725141 234053936 20.93 1.35 0 2.80 0 0.11 0 0.03 0 0.00 0 10.05 0 6346865 0 200 0 196.83 0 1.59 0 0.02 0 1.50 0 0.01 0 153.25 0 0.38 0 95085 0 7066733 0 197632 0 7636 0 2372 0 0 0 709860 0 1189 0 0 0 13358 0 1740161 0 16439 0 1771147 0 87.02 0 6149233 0 36843 1715269 46.556170778710 7066733.0 6346865.0 95085.0 197632.0 7636.0 2372.0 0.0 709860.0 6149233.0 89.8 1.3 2.8 0.1 0.0 0.0 10.0 87.0 100 100 100.00 38 706673300 27.8 21.5 21.2 29.5 0.0 35.7 18.2 smartseq 502204 SRR2049537 SRP059035 SRS951834 SRX1047617 SRA271224 GEO Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. Overall design: We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. GSM1702785: LC-PT-45-Selumetinib_R0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol. Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Illumina HiSeq 2500 cell type;;PDX|source_name;;PDX, LC-PT-45 GEO Accession;;GSM1702785 GSM1702785 LC-PT-45-Selumetinib_R0 1166115000 5830575 2015-06-05 16:18:03 787318884 1166115000 5830575 2 5830575 index:0,count:5830575,average:100,stdev:0|index:1,count:5830575,average:100,stdev:0 GSM1702785_r1 4.75 2.1 0.05 791000958 1071271224 732702057 1008691304 135.43 137.67 4811213 4126689 238.903 3270.250 90 25678 83.64 90.79 5827246 4024015 5827246 4024015 83.59 85.67 5827246 4021672 5827246 3796987 33161304 4.19 3.03 0 6.50 0 0.21 0 0.03 0 0.00 0 17.25 0 4811213 0 200 0 194.11 0 1.47 0 0.01 0 1.50 0 0.01 0 90.47 0 0.64 0 176771 0 5830575 0 378990 0 12035 0 1500 0 0 0 1005827 0 670 0 0 0 13182 0 1950143 0 13081 0 1977076 0 76.02 0 4432223 0 109575 1861770 16.990828199863 5830575.0 4811213.0 176771.0 378990.0 12035.0 1500.0 0.0 1005827.0 4432223.0 82.5 3.0 6.5 0.2 0.0 0.0 17.3 76.0 100 100 100.00 38 583057500 25.1 24.0 23.4 27.5 0.0 35.1 18.0 smartseq 1258536 SRR2063342 SRP059509 SRS961372 SRX1059115 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712023: Human islet polyA RNA-Seq donor 3 CT16; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1174|strain;;none GEO Accession;;GSM1712023 GSM1712023 Human islet polyA RNA-Seq donor 3 CT16 6198759579 41065560 2015-11-10 17:09:03 3080133634 6198759579 41065560 2 41065560 index:0,count:41065560,average:75.48,stdev:1.04|index:1,count:41065560,average:75.46,stdev:1.15 GSM1712023_r1 5.37 2.39 0.09 5038123513 6656085246 4808988715 6391492717 132.11 132.91 37088239 33930105 177.115 747.984 135 378151 87.36 91.59 41279568 32403398 41279568 32403398 81.67 82.38 41279568 30292218 41279568 29145334 152593080 3.03 0.75 0 4.17 0 0.02 0 0.02 0 0.00 0 9.64 0 37089835 0 150 0 148.92 0 1.54 0 0.01 0 1.24 0 0.01 0 262.12 0 1.15 0 306311 0 41065560 0 1711671 0 10053 0 6386 0 0 0 3959286 0 3936 0 0 0 57407 0 11668102 0 23171 0 11752616 0 86.15 0 35378164 0 233448 11710419 50.162858538090 41065560.0 37089835.0 306311.0 1711671.0 10053.0 6386.0 0.0 3959286.0 35378164.0 90.3 0.7 4.2 0.0 0.0 0.0 9.6 86.2 35 76 75.48 7 3099810524 26.0 23.7 24.8 25.5 0.0 32.9 20.9 bulk 1258553 SRR2063343 SRP059509 SRS961371 SRX1059116 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712024: Human islet polyA RNA-Seq donor 3 CT20; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1174|strain;;none GEO Accession;;GSM1712024 GSM1712024 Human islet polyA RNA-Seq donor 3 CT20 6650354175 44062970 2015-11-10 17:09:03 3302387299 6650354175 44062970 2 44062970 index:0,count:44062970,average:75.47,stdev:1.08|index:1,count:44062970,average:75.45,stdev:1.18 GSM1712024_r1 4.81 2.33 0.1 5309579239 6941983023 5059073666 6646064554 130.74 131.37 39265491 35791630 177.434 734.054 127 404431 88.64 93.1 43510099 34805069 43510099 34805069 81.85 82.38 43510099 32138588 43510099 30798850 129328230 2.44 0.66 0 4.27 0 0.02 0 0.01 0 0.00 0 10.85 0 39267198 0 150 0 148.88 0 1.53 0 0.01 0 1.24 0 0.01 0 239.62 0 1.11 0 291376 0 44062970 0 1881985 0 8292 0 5714 0 0 0 4781766 0 4153 0 0 0 57412 0 13035194 0 25620 0 13122379 0 84.84 0 37385213 0 238502 13020145 54.591345145953 44062970.0 39267198.0 291376.0 1881985.0 8292.0 5714.0 0.0 4781766.0 37385213.0 89.1 0.7 4.3 0.0 0.0 0.0 10.9 84.8 35 76 75.47 7 3325617781 26.3 23.7 24.3 25.7 0.0 32.8 20.8 bulk 629044 SRR2063326 SRP059509 SRS961388 SRX1059099 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712007: Human islet polyA RNA-Seq donor 1 CT0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1114|strain;;none GEO Accession;;GSM1712007 GSM1712007 Human islet polyA RNA-Seq donor 1 CT0 5379600483 35665896 2015-11-10 17:09:03 2386664105 5379600483 35665896 2 35665896 index:0,count:35665896,average:75.44,stdev:1.42|index:1,count:35665896,average:75.39,stdev:1.59 GSM1712007_r1 5.83 2.37 0.13 4392473386 6008606537 4150761407 5724012476 136.79 137.9 34330498 32377809 151.743 536.333 119 446075 88.16 93.45 39213981 30267862 39213981 30267862 86.13 87.17 39213981 29568299 39213981 28234361 102500583 2.33 0.21 0 5.44 0 0.04 0 0.03 0 0.00 0 3.67 0 34331312 0 150 0 149.31 0 1.53 0 0.01 0 1.29 0 0.01 0 278.52 0 0.58 0 74122 0 35665896 0 1941690 0 13161 0 11897 0 0 0 1309526 0 4460 0 0 0 73246 0 11710656 0 21787 0 11810149 0 90.81 0 32389622 0 234410 11338480 48.370291369822 35665896.0 34331312.0 74122.0 1941690.0 13161.0 11897.0 0.0 1309526.0 32389622.0 96.3 0.2 5.4 0.0 0.0 0.0 3.7 90.8 35 76 75.44 7 2690795620 25.0 23.8 24.3 26.9 0.0 34.4 23.2 bulk 629052 SRR2063327 SRP059509 SRS961387 SRX1059100 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712008: Human islet polyA RNA-Seq donor 1 CT4; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1114|strain;;none GEO Accession;;GSM1712008 GSM1712008 Human islet polyA RNA-Seq donor 1 CT4 5394548636 35761428 2015-11-10 17:09:03 2389822699 5394548636 35761428 2 35761428 index:0,count:35761428,average:75.45,stdev:1.34|index:1,count:35761428,average:75.40,stdev:1.52 GSM1712008_r1 5.36 2.37 0.16 4396766593 5984328336 4161182570 5711268591 136.11 137.25 34408033 32731164 148.258 484.734 119 471484 86.77 91.82 39187580 29856978 39187580 29856978 85.04 86.11 39187580 29262333 39187580 28001273 130843833 2.98 0.21 0 5.29 0 0.04 0 0.04 0 0.00 0 3.71 0 34408878 0 150 0 149.42 0 1.52 0 0.01 0 1.28 0 0.01 0 311.72 0 0.56 0 75419 0 35761428 0 1892033 0 13698 0 13176 0 0 0 1325676 0 4169 0 0 0 68643 0 10762477 0 20918 0 10856207 0 90.93 0 32516845 0 227865 10462685 45.916156496171 35761428.0 34408878.0 75419.0 1892033.0 13698.0 13176.0 0.0 1325676.0 32516845.0 96.2 0.2 5.3 0.0 0.0 0.0 3.7 90.9 35 76 75.45 7 2698042525 26.1 23.2 23.4 27.3 0.0 34.4 23.2 bulk 629060 SRR2063328 SRP059509 SRS961386 SRX1059101 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712009: Human islet polyA RNA-Seq donor 1 CT8; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1114|strain;;none GEO Accession;;GSM1712009 GSM1712009 Human islet polyA RNA-Seq donor 1 CT8 4643583168 30763785 2015-11-10 17:09:03 2067220337 4643583168 30763785 2 30763785 index:0,count:30763785,average:75.49,stdev:1.14|index:1,count:30763785,average:75.45,stdev:1.35 GSM1712009_r1 4.7 2.3 0.13 4011228528 5474029829 3790152194 5223231500 136.47 137.81 29584100 27134321 179.437 731.015 128 295662 88.1 93.32 34059978 26065544 34059978 26065544 85.83 87.01 34059978 25392245 34059978 24303235 95238027 2.37 0.23 0 5.38 0 0.04 0 0.03 0 0.00 0 3.76 0 29584738 0 150 0 149.41 0 1.57 0 0.01 0 1.30 0 0.00 0 256.36 0 0.60 0 71236 0 30763785 0 1653893 0 11852 0 10087 0 0 0 1157108 0 3751 0 0 0 60336 0 9916953 0 18932 0 9999972 0 90.79 0 27930845 0 232987 10004918 42.941958135003 30763785.0 29584738.0 71236.0 1653893.0 11852.0 10087.0 0.0 1157108.0 27930845.0 96.2 0.2 5.4 0.0 0.0 0.0 3.8 90.8 35 76 75.49 7 2322437339 24.9 24.4 24.7 26.0 0.0 34.4 23.2 bulk 629069 SRR2063329 SRP059509 SRS961385 SRX1059102 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712010: Human islet polyA RNA-Seq donor 1 CT12; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1114|strain;;none GEO Accession;;GSM1712010 GSM1712010 Human islet polyA RNA-Seq donor 1 CT12 4729312142 31351635 2015-11-10 17:09:03 2103346669 4729312142 31351635 2 31351635 index:0,count:31351635,average:75.44,stdev:1.34|index:1,count:31351635,average:75.40,stdev:1.51 GSM1712010_r1 5.2 2.39 0.14 3896022983 5252105854 3698563384 5025146565 134.81 135.87 30138262 28594741 151.197 503.968 128 397824 86.52 91.25 34133933 26075308 34133933 26075308 84.39 85.44 34133933 25433217 34133933 24414356 125385847 3.22 0.22 0 4.99 0 0.04 0 0.03 0 0.00 0 3.80 0 30138983 0 150 0 149.41 0 1.54 0 0.01 0 1.29 0 0.01 0 340.98 0 0.58 0 70142 0 31351635 0 1564623 0 11539 0 10097 0 0 0 1191016 0 3685 0 0 0 57017 0 9312524 0 18156 0 9391382 0 91.14 0 28574360 0 229132 9093872 39.688354311052 31351635.0 30138983.0 70142.0 1564623.0 11539.0 10097.0 0.0 1191016.0 28574360.0 96.1 0.2 5.0 0.0 0.0 0.0 3.8 91.1 35 76 75.44 7 2365259762 26.2 23.3 23.4 27.1 0.0 34.3 23.1 bulk 629125 SRR2063330 SRP059509 SRS961384 SRX1059103 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712011: Human islet polyA RNA-Seq donor 1 CT16; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1114|strain;;none GEO Accession;;GSM1712011 GSM1712011 Human islet polyA RNA-Seq donor 1 CT16 4837267262 32069755 2015-11-10 17:09:03 2147784664 4837267262 32069755 2 32069755 index:0,count:32069755,average:75.44,stdev:1.37|index:1,count:32069755,average:75.39,stdev:1.54 GSM1712011_r1 5.98 2.36 0.14 3916528931 5332453167 3698778771 5078777320 136.15 137.31 30711379 29118045 148.204 498.835 119 425338 87.52 92.83 35222991 26880305 35222991 26880305 85.69 86.79 35222991 26316976 35222991 25131619 99305507 2.54 0.19 0 5.48 0 0.04 0 0.03 0 0.00 0 4.17 0 30712091 0 150 0 149.34 0 1.52 0 0.01 0 1.29 0 0.01 0 276.86 0 0.57 0 61901 0 32069755 0 1756607 0 11282 0 10045 0 0 0 1336337 0 3823 0 0 0 64958 0 10210658 0 18508 0 10297947 0 90.29 0 28955484 0 215286 9898991 45.980653642132 32069755.0 30712091.0 61901.0 1756607.0 11282.0 10045.0 0.0 1336337.0 28955484.0 95.8 0.2 5.5 0.0 0.0 0.0 4.2 90.3 35 76 75.44 7 2419498515 25.5 23.3 23.7 27.4 0.0 34.3 23.1 bulk 629134 SRR2063331 SRP059509 SRS961383 SRX1059104 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712012: Human islet polyA RNA-Seq donor 1 CT20; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1114|strain;;none GEO Accession;;GSM1712012 GSM1712012 Human islet polyA RNA-Seq donor 1 CT20 5909588388 39185609 2015-11-10 17:09:03 2624181725 5909588388 39185609 2 39185609 index:0,count:39185609,average:75.43,stdev:1.45|index:1,count:39185609,average:75.38,stdev:1.61 GSM1712012_r1 5.18 2.39 0.15 4714776187 6384057600 4462673172 6091510319 135.41 136.5 37475714 35688317 145.124 476.055 118 526871 86.86 91.9 42657624 32552162 42657624 32552162 84.69 85.74 42657624 31738915 42657624 30370789 137816378 2.92 0.24 0 5.25 0 0.04 0 0.03 0 0.00 0 4.29 0 37476580 0 150 0 149.37 0 1.52 0 0.01 0 1.28 0 0.01 0 323.55 0 0.57 0 92486 0 39185609 0 2056327 0 14577 0 13662 0 0 0 1680790 0 4524 0 0 0 71738 0 11848094 0 23067 0 11947423 0 90.39 0 35420253 0 239504 11364364 47.449579130202 39185609.0 37476580.0 92486.0 2056327.0 14577.0 13662.0 0.0 1680790.0 35420253.0 95.6 0.2 5.2 0.0 0.0 0.0 4.3 90.4 35 76 75.43 7 2955633609 26.0 23.2 23.8 27.0 0.0 34.4 23.1 bulk 629140 SRR2063332 SRP059509 SRS961382 SRX1059105 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712013: Human islet polyA RNA-Seq donor 2 CT0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1121|strain;;none GEO Accession;;GSM1712013 GSM1712013 Human islet polyA RNA-Seq donor 2 CT0 5329814764 35350014 2015-11-10 17:09:03 2373573172 5329814764 35350014 2 35350014 index:0,count:35350014,average:75.41,stdev:1.51|index:1,count:35350014,average:75.36,stdev:1.67 GSM1712013_r1 3.33 2.49 0.11 4188889590 5522343645 3989554191 5298307044 131.83 132.8 33672710 32118322 142.221 444.562 118 496240 86.98 91.45 38029839 29289161 38029839 29289161 83.31 84.32 38029839 28053781 38029839 27005876 127597317 3.05 0.21 0 4.65 0 0.03 0 0.03 0 0.00 0 4.69 0 33673480 0 150 0 149.34 0 1.52 0 0.01 0 1.30 0 0.01 0 305.91 0 0.56 0 74371 0 35350014 0 1644846 0 10614 0 9303 0 0 0 1656617 0 4088 0 0 0 59292 0 10654180 0 22205 0 10739765 0 90.60 0 32028634 0 238606 10056698 42.147716318953 35350014.0 33673480.0 74371.0 1644846.0 10614.0 9303.0 0.0 1656617.0 32028634.0 95.3 0.2 4.7 0.0 0.0 0.0 4.7 90.6 35 76 75.41 7 2665683619 26.3 23.6 23.1 27.0 0.0 34.2 22.8 bulk 629148 SRR2063333 SRP059509 SRS961381 SRX1059106 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712014: Human islet polyA RNA-Seq donor 2 CT4; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1121|strain;;none GEO Accession;;GSM1712014 GSM1712014 Human islet polyA RNA-Seq donor 2 CT4 4797646773 31799756 2015-11-10 17:09:03 2134613380 4797646773 31799756 2 31799756 index:0,count:31799756,average:75.45,stdev:1.27|index:1,count:31799756,average:75.42,stdev:1.45 GSM1712014_r1 4.06 2.46 0.12 3959376394 5257623786 3781572188 5048954651 132.79 133.51 30459034 28868638 153.985 484.462 128 400600 87.16 91.36 33726624 26550176 33726624 26550176 83.32 84.07 33726624 25378288 33726624 24430519 121596614 3.07 0.22 0 4.40 0 0.03 0 0.02 0 0.00 0 4.16 0 30459780 0 150 0 149.44 0 1.53 0 0.01 0 1.30 0 0.01 0 285.48 0 0.57 0 69231 0 31799756 0 1398767 0 9935 0 7930 0 0 0 1322111 0 3466 0 0 0 53237 0 9513133 0 20040 0 9589876 0 91.39 0 29061013 0 230287 9330010 40.514705563058 31799756.0 30459780.0 69231.0 1398767.0 9935.0 7930.0 0.0 1322111.0 29061013.0 95.8 0.2 4.4 0.0 0.0 0.0 4.2 91.4 35 76 75.45 7 2399427590 26.1 23.6 23.5 26.8 0.0 34.3 23.1 bulk 629158 SRR2063334 SRP059509 SRS961380 SRX1059107 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712015: Human islet polyA RNA-Seq donor 2 CT8; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1121|strain;;none GEO Accession;;GSM1712015 GSM1712015 Human islet polyA RNA-Seq donor 2 CT8 5024833928 33331542 2015-11-10 17:09:03 2233798128 5024833928 33331542 2 33331542 index:0,count:33331542,average:75.40,stdev:1.55|index:1,count:33331542,average:75.36,stdev:1.70 GSM1712015_r1 3.64 2.47 0.12 3936104881 5178817852 3743681237 4961007658 131.57 132.52 31817183 30472943 140.318 412.848 118 482940 86.31 90.89 35978651 27463420 35978651 27463420 82.95 83.98 35978651 26393986 35978651 25376405 128354770 3.26 0.21 0 4.80 0 0.03 0 0.03 0 0.00 0 4.48 0 31817972 0 150 0 149.36 0 1.52 0 0.01 0 1.29 0 0.01 0 304.55 0 0.55 0 70621 0 33331542 0 1600828 0 10018 0 9054 0 0 0 1494498 0 3605 0 0 0 52186 0 9582189 0 20842 0 9658822 0 90.66 0 30217144 0 228590 9035429 39.526790323286 33331542.0 31817972.0 70621.0 1600828.0 10018.0 9054.0 0.0 1494498.0 30217144.0 95.5 0.2 4.8 0.0 0.0 0.0 4.5 90.7 35 76 75.40 7 2513041737 26.8 23.0 22.7 27.4 0.0 34.3 23.0 bulk 629166 SRR2063335 SRP059509 SRS961379 SRX1059108 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712016: Human islet polyA RNA-Seq donor 2 CT12; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1121|strain;;none GEO Accession;;GSM1712016 GSM1712016 Human islet polyA RNA-Seq donor 2 CT12 3529808475 23409592 2015-11-10 17:09:03 1577825166 3529808475 23409592 2 23409592 index:0,count:23409592,average:75.41,stdev:1.47|index:1,count:23409592,average:75.37,stdev:1.63 GSM1712016_r1 4.06 2.48 0.13 2821012880 3701537498 2689795924 3551051403 131.21 132.02 22410967 21416002 143.242 437.644 118 324884 84.85 89.11 25016977 19016110 25016977 19016110 81.81 82.65 25016977 18333756 25016977 17637771 114290046 4.05 0.24 0 4.58 0 0.03 0 0.03 0 0.00 0 4.20 0 22411516 0 150 0 149.37 0 1.51 0 0.01 0 1.29 0 0.01 0 221.78 0 0.58 0 55308 0 23409592 0 1072212 0 7902 0 7420 0 0 0 982754 0 2655 0 0 0 38447 0 6718505 0 14668 0 6774275 0 91.16 0 21339304 0 212988 6432554 30.201485529701 23409592.0 22411516.0 55308.0 1072212.0 7902.0 7420.0 0.0 982754.0 21339304.0 95.7 0.2 4.6 0.0 0.0 0.0 4.2 91.2 35 76 75.41 7 1765378661 26.7 22.8 23.0 27.5 0.0 34.3 23.0 bulk 629174 SRR2063336 SRP059509 SRS961378 SRX1059109 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712017: Human islet polyA RNA-Seq donor 2 CT16; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1121|strain;;none GEO Accession;;GSM1712017 GSM1712017 Human islet polyA RNA-Seq donor 2 CT16 4943690939 32785954 2015-11-10 17:09:03 2204407235 4943690939 32785954 2 32785954 index:0,count:32785954,average:75.41,stdev:1.44|index:1,count:32785954,average:75.37,stdev:1.60 GSM1712017_r1 3.62 2.59 0.13 3960189632 5168398319 3792512304 4978917884 130.51 131.28 31465957 30090315 142.530 431.437 118 461333 84.57 88.42 34758786 26612888 34758786 26612888 81.39 82.2 34758786 25611398 34758786 24739050 171159371 4.32 0.22 0 4.18 0 0.03 0 0.03 0 0.00 0 3.96 0 31466725 0 150 0 149.39 0 1.52 0 0.01 0 1.29 0 0.01 0 287.18 0 0.57 0 70589 0 32785954 0 1369858 0 11068 0 9835 0 0 0 1298326 0 3570 0 0 0 52548 0 9184058 0 20185 0 9260361 0 91.80 0 30096867 0 229095 8712759 38.031205395142 32785954.0 31466725.0 70589.0 1369858.0 11068.0 9835.0 0.0 1298326.0 30096867.0 96.0 0.2 4.2 0.0 0.0 0.0 4.0 91.8 35 76 75.41 7 2472539444 27.0 22.8 22.4 27.8 0.0 34.3 23.1 bulk 629182 SRR2063337 SRP059509 SRS961377 SRX1059110 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712018: Human islet polyA RNA-Seq donor 2 CT20; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1121|strain;;none GEO Accession;;GSM1712018 GSM1712018 Human islet polyA RNA-Seq donor 2 CT20 4695187198 31130319 2015-11-10 17:09:03 2089407277 4695187198 31130319 2 31130319 index:0,count:31130319,average:75.43,stdev:1.37|index:1,count:31130319,average:75.39,stdev:1.54 GSM1712018_r1 3.61 2.57 0.11 3786542338 4997653419 3617634725 4807693113 131.98 132.9 29779971 28316778 146.954 467.928 118 408672 86.5 90.65 33270221 25761167 33270221 25761167 82.68 83.58 33270221 24624036 33270221 23752519 127923656 3.38 0.20 0 4.38 0 0.03 0 0.02 0 0.00 0 4.28 0 29780686 0 150 0 149.39 0 1.53 0 0.01 0 1.31 0 0.01 0 302.89 0 0.56 0 62211 0 31130319 0 1363250 0 9666 0 7757 0 0 0 1332210 0 3479 0 0 0 53194 0 9326698 0 19327 0 9402698 0 91.29 0 28417436 0 234035 8891844 37.993650522358 31130319.0 29780686.0 62211.0 1363250.0 9666.0 7757.0 0.0 1332210.0 28417436.0 95.7 0.2 4.4 0.0 0.0 0.0 4.3 91.3 35 76 75.43 7 2348248803 26.2 23.5 23.2 27.1 0.0 34.3 23.0 bulk 629190 SRR2063338 SRP059509 SRS961376 SRX1059111 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712019: Human islet polyA RNA-Seq donor 3 CT0; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1174|strain;;none GEO Accession;;GSM1712019 GSM1712019 Human islet polyA RNA-Seq donor 3 CT0 6260623011 41482174 2015-11-10 17:09:03 3137665768 6260623011 41482174 2 41482174 index:0,count:41482174,average:75.48,stdev:1.07|index:1,count:41482174,average:75.45,stdev:1.16 GSM1712019_r1 5.58 2.4 0.09 4693135970 6252614862 4470900520 5997053241 133.23 134.14 34764969 32127890 173.438 700.780 135 375001 86.7 91.09 38985996 30144233 38985996 30144233 81.19 82.01 38985996 28227297 38985996 27140640 152114261 3.24 1.21 0 4.03 0 0.03 0 0.02 0 0.00 0 16.14 0 34766588 0 150 0 148.96 0 1.54 0 0.01 0 1.25 0 0.01 0 174.25 0 1.14 0 500353 0 41482174 0 1672418 0 11012 0 8517 0 0 0 6696057 0 3544 0 0 0 53632 0 10529326 0 22140 0 10608642 0 79.78 0 33094170 0 231202 10562703 45.686036452972 41482174.0 34766588.0 500353.0 1672418.0 11012.0 8517.0 0.0 6696057.0 33094170.0 83.8 1.2 4.0 0.0 0.0 0.0 16.1 79.8 35 76 75.48 7 3130941019 26.2 23.7 24.9 25.2 0.0 32.7 20.6 bulk 629198 SRR2063339 SRP059509 SRS961375 SRX1059112 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712020: Human islet polyA RNA-Seq donor 3 CT4; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1174|strain;;none GEO Accession;;GSM1712020 GSM1712020 Human islet polyA RNA-Seq donor 3 CT4 6473188134 42882394 2015-11-10 17:09:03 3216555938 6473188134 42882394 2 42882394 index:0,count:42882394,average:75.49,stdev:1.00|index:1,count:42882394,average:75.47,stdev:1.11 GSM1712020_r1 5.18 2.36 0.1 5193492790 6833019285 4960650981 6551682252 131.57 132.07 37947152 34594258 180.436 748.796 135 398612 88.74 92.96 41559161 33676695 41559161 33676695 81.88 82.28 41559161 31073481 41559161 29805666 128054098 2.47 0.73 0 4.02 0 0.02 0 0.01 0 0.00 0 11.47 0 37948878 0 150 0 148.92 0 1.52 0 0.01 0 1.24 0 0.01 0 228.71 0 1.12 0 313066 0 42882394 0 1723562 0 8167 0 5867 0 0 0 4919482 0 3973 0 0 0 57934 0 12609232 0 25493 0 12696632 0 84.48 0 36225316 0 229297 12737074 55.548367401231 42882394.0 37948878.0 313066.0 1723562.0 8167.0 5867.0 0.0 4919482.0 36225316.0 88.5 0.7 4.0 0.0 0.0 0.0 11.5 84.5 35 76 75.49 7 3236997429 26.1 23.8 24.7 25.5 0.0 32.8 20.8 bulk 629253 SRR2063340 SRP059509 SRS961374 SRX1059113 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712021: Human islet polyA RNA-Seq donor 3 CT8; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1174|strain;;none GEO Accession;;GSM1712021 GSM1712021 Human islet polyA RNA-Seq donor 3 CT8 6258528953 41457760 2015-11-10 17:09:03 3107169898 6258528953 41457760 2 41457760 index:0,count:41457760,average:75.49,stdev:0.97|index:1,count:41457760,average:75.47,stdev:1.08 GSM1712021_r1 5.81 2.46 0.1 5121593011 6826726866 4902983259 6570754789 133.29 134.02 37117604 33840396 184.484 777.296 135 353743 86.78 90.71 40794894 32213420 40794894 32213420 82.0 82.65 40794894 30439605 40794894 29350399 172535115 3.37 0.53 0 3.87 0 0.03 0 0.02 0 0.00 0 10.42 0 37119211 0 150 0 149.01 0 1.56 0 0.01 0 1.25 0 0.01 0 209.32 0 1.12 0 221378 0 41457760 0 1605658 0 11237 0 6312 0 0 0 4321000 0 3746 0 0 0 58113 0 11065426 0 23376 0 11150661 0 85.66 0 35513553 0 243109 11221509 46.158344610854 41457760.0 37119211.0 221378.0 1605658.0 11237.0 6312.0 0.0 4321000.0 35513553.0 89.5 0.5 3.9 0.0 0.0 0.0 10.4 85.7 35 76 75.49 7 3129701189 26.3 23.6 24.4 25.7 0.0 32.8 20.8 bulk 629262 SRR2063341 SRP059509 SRS961373 SRX1059114 SRA272961 GEO Genome-wide Circadian Control of Transcription at Active Enhancers Regulates Insulin Secretion and Diabetes Risk The molecular clock is a transcriptional oscillator present in brain and peripheral cells that coordinates behavior and physiology with the solar cycle. Here we reveal that the clock gates insulin secretion through genome-wide transcriptional control of the pancreatic exocyst across species. Clock transcription factors bind to unique enhancer sites in cycling genes in beta cells that diverge from those in liver, revealing the dynamics of inter-tissue clock control of genomic and physiologic processes important in glucose homeostasis. Overall design: Transcriptome profiling in mouse and human islets at serial 4-hour time intervals by polyA RNA-Seq GSM1712022: Human islet polyA RNA-Seq donor 3 CT12; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Mouse islets were isolated by perfusing the pancreatic bile duct with collagenase Illumina stranded LT kit for RNA-Seq NextSeq 500 cell type;;Pancreatic islet cells|source_name;;Human donor IIDP ID 1174|strain;;none GEO Accession;;GSM1712022 GSM1712022 Human islet polyA RNA-Seq donor 3 CT12 7438505190 49278200 2015-11-10 17:09:03 3712472538 7438505190 49278200 2 49278200 index:0,count:49278200,average:75.49,stdev:1.00|index:1,count:49278200,average:75.46,stdev:1.11 GSM1712022_r1 5.51 2.5 0.1 6012227873 8002735745 5754594405 7701058714 133.11 133.82 43828563 40001051 180.827 770.926 135 437944 87.08 91.04 48199004 38169701 48199004 38169701 82.53 83.18 48199004 36171451 48199004 34874367 195469675 3.25 0.68 0 3.86 0 0.03 0 0.02 0 0.00 0 11.01 0 43830484 0 150 0 148.92 0 1.54 0 0.01 0 1.25 0 0.01 0 230.09 0 1.17 0 334227 0 49278200 0 1903317 0 12652 0 7622 0 0 0 5427442 0 4426 0 0 0 69302 0 13262908 0 28401 0 13365037 0 85.08 0 41927167 0 252845 13402341 53.006153967846 49278200.0 43830484.0 334227.0 1903317.0 12652.0 7622.0 0.0 5427442.0 41927167.0 88.9 0.7 3.9 0.0 0.0 0.0 11.0 85.1 35 76 75.49 7 3719822802 26.5 23.5 24.4 25.7 0.0 32.7 20.7 bulk 1050456 SRR2088075 SRP060416 SRS980413 SRX1082044 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810510: T74_P1_A9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810510 GSM1810510 T74_P1_A9_ILC1 40278616 936712 2016-01-28 01:00:06 46422924 40278616 936712 1 936712 index:0,count:936712,average:43,stdev:0 GSM1810510_r1 3.25 9.87 0.23 26835977 32561194 20251403 25660431 121.33 126.71 0 0 0 0 0 0 53.79 72.94 1155151 358275 1155151 358275 61.14 70.33 1155151 407240 1155151 345486 4561917 17.00 6.43 0 18.67 0 1.22 0 0.31 0 0.00 0 27.36 0 666095 0 43 0 41.23 0 1.23 0 0.01 0 1.09 0 0.00 0 168.61 0 0.44 0 60238 0 936712 0 174894 0 11451 0 2902 0 0 0 256264 0 8 0 0 0 104 0 13143 0 289 0 13544 0 52.44 0 491201 0 2742 15518 5.659372720642 936712.0 666095.0 60238.0 174894.0 11451.0 2902.0 0.0 256264.0 491201.0 71.1 6.4 18.7 1.2 0.3 0.0 27.4 52.4 43 43 43.00 38 40278616 26.7 20.2 21.2 31.9 0.0 34.3 22.1 smartseq 1050472 SRR2088076 SRP060416 SRS980412 SRX1082045 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810511: T74_P1_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810511 GSM1810511 T74_P1_B4_NK 223422152 5195864 2016-01-28 01:00:06 238793144 223422152 5195864 1 5195864 index:0,count:5195864,average:43,stdev:0 GSM1810511_r1 6.13 6.07 0.25 160540205 210914226 128856985 173713442 131.38 134.81 0 0 0 0 0 0 64.2 81.04 6032934 2499913 6032934 2499913 71.51 77.34 6032934 2784588 6032934 2385821 17957961 11.19 5.33 0 15.58 0 0.98 0 0.47 0 0.00 0 23.61 0 3894189 0 43 0 41.77 0 1.17 0 0.01 0 1.16 0 0.00 0 415.67 0 0.32 0 276721 0 5195864 0 809332 0 50777 0 24208 0 0 0 1226690 0 45 0 0 0 857 0 117385 0 1596 0 119883 0 59.37 0 3084857 0 6976 137084 19.650802752294 5195864.0 3894189.0 276721.0 809332.0 50777.0 24208.0 0.0 1226690.0 3084857.0 74.9 5.3 15.6 1.0 0.5 0.0 23.6 59.4 43 43 43.00 38 223422152 26.3 22.2 22.3 29.2 0.0 36.1 24.6 smartseq 1050488 SRR2088077 SRP060416 SRS980411 SRX1082046 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810512: T74_P1_B7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810512 GSM1810512 T74_P1_B7_ILC2 148565473 3455011 2016-01-28 01:00:06 160445145 148565473 3455011 1 3455011 index:0,count:3455011,average:43,stdev:0 GSM1810512_r1 5.19 8.24 0.29 95231366 119680783 72868298 94885063 125.67 130.21 0 0 0 0 0 0 58.77 78.27 3896420 1374760 3896420 1374760 66.45 75.2 3896420 1554204 3896420 1320786 13533612 14.21 6.95 0 16.86 0 1.09 0 0.37 0 0.00 0 30.84 0 2339078 0 43 0 41.49 0 1.15 0 0.01 0 1.13 0 0.00 0 222.11 0 0.32 0 240103 0 3455011 0 582607 0 37618 0 12630 0 0 0 1065685 0 22 0 0 0 483 0 51892 0 993 0 53390 0 50.84 0 1756471 0 3264 61965 18.984375000000 3455011.0 2339078.0 240103.0 582607.0 37618.0 12630.0 0.0 1065685.0 1756471.0 67.7 6.9 16.9 1.1 0.4 0.0 30.8 50.8 43 43 43.00 38 148565473 26.6 21.5 21.5 30.5 0.0 35.9 24.1 smartseq 1050504 SRR2088078 SRP060416 SRS980409 SRX1082047 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810513: T74_P1_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810513 GSM1810513 T74_P1_B9_NK 150176855 3492485 2016-01-28 01:00:06 161619993 150176855 3492485 1 3492485 index:0,count:3492485,average:43,stdev:0 GSM1810513_r1 3.54 7.45 0.43 98941624 121634312 79707610 101553535 122.94 127.41 0 0 0 0 0 0 58.52 74.02 3790530 1419930 3790530 1419930 63.18 70.4 3790530 1533003 3790530 1350428 16189565 16.36 6.51 0 14.55 0 1.12 0 0.41 0 0.00 0 28.99 0 2426386 0 43 0 41.55 0 1.22 0 0.00 0 1.18 0 0.00 0 292.39 0 0.32 0 227314 0 3492485 0 508177 0 39131 0 14322 0 0 0 1012646 0 40 0 0 0 419 0 55502 0 1076 0 57037 0 54.92 0 1918209 0 3471 65325 18.820224719101 3492485.0 2426386.0 227314.0 508177.0 39131.0 14322.0 0.0 1012646.0 1918209.0 69.5 6.5 14.6 1.1 0.4 0.0 29.0 54.9 43 43 43.00 38 150176855 26.9 21.4 21.5 30.2 0.0 36.0 24.2 smartseq 1050520 SRR2088079 SRP060416 SRS980410 SRX1082048 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810514: T74_P1_D10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810514 GSM1810514 T74_P1_D10_ILC2 46380918 1078626 2016-01-28 01:00:06 50853978 46380918 1078626 1 1078626 index:0,count:1078626,average:43,stdev:0 GSM1810514_r1 4.78 6.53 0.26 36439726 48454566 30353237 41083530 132.97 135.35 0 0 0 0 0 0 67.9 82.33 1267648 597512 1267648 597512 74.28 79.08 1267648 653633 1267648 573962 3799009 10.43 3.80 0 14.29 0 1.10 0 0.39 0 0.00 0 16.93 0 879939 0 43 0 41.82 0 1.20 0 0.01 0 1.12 0 0.00 0 242.69 0 0.30 0 40978 0 1078626 0 154169 0 11868 0 4156 0 0 0 182663 0 5 0 0 0 125 0 23813 0 324 0 24267 0 67.29 0 725770 0 2759 27924 10.121058354476 1078626.0 879939.0 40978.0 154169.0 11868.0 4156.0 0.0 182663.0 725770.0 81.6 3.8 14.3 1.1 0.4 0.0 16.9 67.3 43 43 43.00 38 46380918 27.2 21.7 21.8 29.3 0.0 36.1 24.9 smartseq 1050632 SRR2088080 SRP060416 SRS980408 SRX1082049 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810515: T74_P1_E1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810515 GSM1810515 T74_P1_E1_ILC3 72459085 1685095 2016-01-28 01:00:06 80618089 72459085 1685095 1 1685095 index:0,count:1685095,average:43,stdev:0 GSM1810515_r1 12.98 5.08 0.25 57376723 79829454 46197665 65243349 139.13 141.23 0 0 0 0 0 0 65.93 82.52 2060256 907599 2060256 907599 75.13 78.72 2060256 1034295 2060256 865750 5098302 8.89 3.84 0 16.43 0 0.83 0 0.41 0 0.00 0 17.06 0 1376637 0 43 0 42.00 0 1.11 0 0.01 0 1.17 0 0.00 0 224.68 0 0.35 0 64649 0 1685095 0 276813 0 14064 0 6937 0 0 0 287457 0 17 0 0 0 219 0 38574 0 353 0 39163 0 65.27 0 1099824 0 7613 45260 5.945093918298 1685095.0 1376637.0 64649.0 276813.0 14064.0 6937.0 0.0 287457.0 1099824.0 81.7 3.8 16.4 0.8 0.4 0.0 17.1 65.3 43 43 43.00 38 72459085 26.8 22.3 22.6 28.4 0.0 35.7 24.4 smartseq 1050648 SRR2088081 SRP060416 SRS980407 SRX1082050 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810516: T74_P1_E2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810516 GSM1810516 T74_P1_E2_ILC3 24520105 570235 2016-01-28 01:00:06 28477076 24520105 570235 1 570235 index:0,count:570235,average:43,stdev:0 GSM1810516_r1 6.46 8.59 0.27 16674012 20913919 12875443 16732628 125.43 129.96 0 0 0 0 0 0 56.86 75.07 681599 233391 681599 233391 64.15 71.99 681599 263350 681599 223822 2511451 15.06 6.28 0 17.47 0 1.03 0 0.25 0 0.00 0 26.73 0 410497 0 43 0 41.41 0 1.15 0 0.01 0 1.16 0 0.00 0 21.38 0 0.46 0 35807 0 570235 0 99597 0 5866 0 1443 0 0 0 152429 0 1 0 0 0 56 0 10027 0 173 0 10257 0 54.52 0 310900 0 3594 11244 3.128547579299 570235.0 410497.0 35807.0 99597.0 5866.0 1443.0 0.0 152429.0 310900.0 72.0 6.3 17.5 1.0 0.3 0.0 26.7 54.5 43 43 43.00 38 24520105 26.4 20.6 21.8 31.2 0.0 34.2 22.1 smartseq 1050664 SRR2088082 SRP060416 SRS980405 SRX1082051 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810517: T74_P1_E3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810517 GSM1810517 T74_P1_E3_ILC3 94234156 2191492 2016-01-28 01:00:06 103805070 94234156 2191492 1 2191492 index:0,count:2191492,average:43,stdev:0 GSM1810517_r1 6.65 6.67 0.26 66321257 85242528 53194479 70228759 128.53 132.02 0 0 0 0 0 0 58.37 73.83 2505178 940004 2505178 940004 65.09 70.58 2505178 1048185 2505178 898649 10295318 15.52 5.74 0 15.39 0 1.00 0 0.58 0 0.00 0 24.94 0 1610454 0 43 0 41.78 0 1.14 0 0.01 0 1.13 0 0.00 0 225.41 0 0.34 0 125870 0 2191492 0 337302 0 21884 0 12648 0 0 0 546506 0 11 0 0 0 261 0 40615 0 595 0 41482 0 58.10 0 1273152 0 6041 46295 7.663466313524 2191492.0 1610454.0 125870.0 337302.0 21884.0 12648.0 0.0 546506.0 1273152.0 73.5 5.7 15.4 1.0 0.6 0.0 24.9 58.1 43 43 43.00 38 94234156 26.6 21.7 21.8 29.9 0.0 35.7 24.2 smartseq 1050680 SRR2088083 SRP060416 SRS980406 SRX1082052 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810518: T74_P1_E4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810518 GSM1810518 T74_P1_E4_ILC3 138811224 3228168 2016-01-28 01:00:06 150746373 138811224 3228168 1 3228168 index:0,count:3228168,average:43,stdev:0 GSM1810518_r1 3.05 8.76 0.23 81774701 99105404 64497657 81295173 121.19 126.04 0 0 0 0 0 0 53.25 69.02 3259272 1072743 3259272 1072743 58.69 66.2 3259272 1182329 3259272 1029012 15593815 19.07 8.19 0 14.25 0 1.21 0 0.37 0 0.00 0 36.01 0 2014434 0 43 0 41.50 0 1.22 0 0.01 0 1.14 0 0.00 0 237.17 0 0.33 0 264295 0 3228168 0 460104 0 39070 0 12085 0 0 0 1162579 0 0 0 0 0 238 0 40303 0 1030 0 41571 0 48.15 0 1554330 0 3766 48424 12.858204992034 3228168.0 2014434.0 264295.0 460104.0 39070.0 12085.0 0.0 1162579.0 1554330.0 62.4 8.2 14.3 1.2 0.4 0.0 36.0 48.1 43 43 43.00 38 138811224 26.1 21.7 21.7 30.5 0.0 35.8 24.0 smartseq 1050696 SRR2088084 SRP060416 SRS980404 SRX1082053 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810519: T74_P1_E5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810519 GSM1810519 T74_P1_E5_ILC3 153838563 3577641 2016-01-28 01:00:06 165761370 153838563 3577641 1 3577641 index:0,count:3577641,average:43,stdev:0 GSM1810519_r1 8.46 6.68 0.19 108386296 143449692 86700894 117635223 132.35 135.68 0 0 0 0 0 0 64.02 81.1 4027143 1683269 4027143 1683269 71.2 77.07 4027143 1872237 4027143 1599669 12824153 11.83 5.76 0 15.48 0 0.87 0 0.38 0 0.00 0 25.26 0 2629369 0 43 0 41.77 0 1.14 0 0.01 0 1.13 0 0.00 0 299.52 0 0.31 0 206204 0 3577641 0 553806 0 30947 0 13537 0 0 0 903788 0 14 0 0 0 398 0 74147 0 1066 0 75625 0 58.01 0 2075563 0 6697 87328 13.039868597880 3577641.0 2629369.0 206204.0 553806.0 30947.0 13537.0 0.0 903788.0 2075563.0 73.5 5.8 15.5 0.9 0.4 0.0 25.3 58.0 43 43 43.00 38 153838563 26.4 22.1 22.0 29.5 0.0 36.1 24.7 smartseq 1050714 SRR2088085 SRP060416 SRS980403 SRX1082054 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810520: T74_P1_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810520 GSM1810520 T74_P1_E6_NK 77916172 1812004 2016-01-28 01:00:06 86116110 77916172 1812004 1 1812004 index:0,count:1812004,average:43,stdev:0 GSM1810520_r1 7.35 5.83 0.14 60398888 80725111 49474923 67704014 133.65 136.85 0 0 0 0 0 0 64.18 79.1 2165305 933738 2165305 933738 71.37 75.82 2165305 1038453 2165305 894934 6779611 11.22 4.14 0 15.15 0 0.92 0 0.43 0 0.00 0 18.36 0 1454955 0 43 0 41.91 0 1.14 0 0.01 0 1.15 0 0.00 0 250.89 0 0.34 0 75015 0 1812004 0 274541 0 16739 0 7711 0 0 0 332599 0 39 0 0 0 257 0 44148 0 523 0 44967 0 65.14 0 1180414 0 6768 52046 7.690011820331 1812004.0 1454955.0 75015.0 274541.0 16739.0 7711.0 0.0 332599.0 1180414.0 80.3 4.1 15.2 0.9 0.4 0.0 18.4 65.1 43 43 43.00 38 77916172 26.5 22.3 22.6 28.6 0.0 35.8 24.4 smartseq 1050730 SRR2088086 SRP060416 SRS980402 SRX1082055 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810521: T74_P1_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810521 GSM1810521 T74_P1_E7_NK 28423774 661018 2016-01-28 01:00:06 32408146 28423774 661018 1 661018 index:0,count:661018,average:43,stdev:0 GSM1810521_r1 4.24 7.49 0.28 19580106 24592597 15414616 20080735 125.6 130.27 0 0 0 0 0 0 58.57 75.75 775317 281350 775317 281350 64.77 72.35 775317 311123 775317 268725 3044910 15.55 5.87 0 16.48 0 1.08 0 0.40 0 0.00 0 25.85 0 480355 0 43 0 41.50 0 1.21 0 0.01 0 1.20 0 0.00 0 158.64 0 0.40 0 38788 0 661018 0 108926 0 7131 0 2662 0 0 0 170870 0 2 0 0 0 90 0 12786 0 190 0 13068 0 56.19 0 371429 0 3278 14818 4.520439292251 661018.0 480355.0 38788.0 108926.0 7131.0 2662.0 0.0 170870.0 371429.0 72.7 5.9 16.5 1.1 0.4 0.0 25.8 56.2 43 43 43.00 38 28423774 27.1 20.9 21.6 30.3 0.0 34.9 22.9 smartseq 1050746 SRR2088087 SRP060416 SRS980400 SRX1082056 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810522: T74_P1_F1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810522 GSM1810522 T74_P1_F1_ILC3 77803254 1809378 2016-01-28 01:00:06 84835443 77803254 1809378 1 1809378 index:0,count:1809378,average:43,stdev:0 GSM1810522_r1 6.24 5.87 0.21 56138090 73643598 46518918 62565175 131.18 134.49 0 0 0 0 0 0 61.58 75.19 2020295 835790 2020295 835790 67.3 71.86 2020295 913380 2020295 798835 7202688 12.83 5.30 0 13.58 0 1.03 0 0.45 0 0.00 0 23.51 0 1357232 0 43 0 41.85 0 1.19 0 0.01 0 1.16 0 0.00 0 197.39 0 0.30 0 95827 0 1809378 0 245637 0 18649 0 8194 0 0 0 425303 0 13 0 0 0 284 0 35511 0 556 0 36364 0 61.44 0 1111595 0 5704 40146 7.038218793829 1809378.0 1357232.0 95827.0 245637.0 18649.0 8194.0 0.0 425303.0 1111595.0 75.0 5.3 13.6 1.0 0.5 0.0 23.5 61.4 43 43 43.00 38 77803254 26.4 22.2 22.2 29.2 0.0 36.2 25.0 smartseq 1050762 SRR2088088 SRP060416 SRS980401 SRX1082057 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810523: T74_P1_F2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810523 GSM1810523 T74_P1_F2_ILC3 200626992 4665744 2016-01-28 01:00:06 216559808 200626992 4665744 1 4665744 index:0,count:4665744,average:43,stdev:0 GSM1810523_r1 7.64 6.54 0.15 140652051 184525898 111764904 150947409 131.19 135.06 0 0 0 0 0 0 63.31 80.73 5347568 2161376 5347568 2161376 71.28 77.33 5347568 2433731 5347568 2070274 16218962 11.53 5.82 0 15.79 0 0.88 0 0.35 0 0.00 0 25.60 0 3414098 0 43 0 41.74 0 1.16 0 0.01 0 1.13 0 0.00 0 305.39 0 0.32 0 271560 0 4665744 0 736757 0 41220 0 16113 0 0 0 1194313 0 43 0 0 0 622 0 104102 0 1416 0 106183 0 57.38 0 2677341 0 7865 120329 15.299300699301 4665744.0 3414098.0 271560.0 736757.0 41220.0 16113.0 0.0 1194313.0 2677341.0 73.2 5.8 15.8 0.9 0.3 0.0 25.6 57.4 43 43 43.00 38 200626992 26.3 22.2 22.1 29.4 0.0 36.0 24.6 smartseq 1050776 SRR2088089 SRP060416 SRS980399 SRX1082058 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810524: T74_P1_F3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810524 GSM1810524 T74_P1_F3_ILC3 178910100 4160700 2016-01-28 01:00:06 192333904 178910100 4160700 1 4160700 index:0,count:4160700,average:43,stdev:0 GSM1810524_r1 8.18 6.41 0.18 131326237 174121554 106873311 145280707 132.59 135.94 0 0 0 0 0 0 65.37 81.32 4794136 2078339 4794136 2078339 71.74 77.2 4794136 2280692 4794136 1972990 15149019 11.54 5.05 0 14.98 0 0.84 0 0.37 0 0.00 0 22.38 0 3179157 0 43 0 41.82 0 1.12 0 0.01 0 1.20 0 0.00 0 427.96 0 0.31 0 210306 0 4160700 0 623360 0 34943 0 15411 0 0 0 931189 0 88 0 0 0 640 0 99173 0 1303 0 101204 0 61.43 0 2555797 0 8769 114213 13.024632227164 4160700.0 3179157.0 210306.0 623360.0 34943.0 15411.0 0.0 931189.0 2555797.0 76.4 5.1 15.0 0.8 0.4 0.0 22.4 61.4 43 43 43.00 38 178910100 26.5 22.0 22.0 29.4 0.0 36.1 24.8 smartseq 1050888 SRR2088090 SRP060416 SRS980397 SRX1082059 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810525: T74_P1_F4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810525 GSM1810525 T74_P1_F4_ILC3 179407739 4172273 2016-01-28 01:00:06 191431628 179407739 4172273 1 4172273 index:0,count:4172273,average:43,stdev:0 GSM1810525_r1 10.95 6.43 0.21 132156860 177277519 107653501 147624924 134.14 137.13 0 0 0 0 0 0 62.85 78.1 4777004 2010561 4777004 2010561 69.71 74.53 4777004 2229974 4777004 1918789 17098321 12.94 4.98 0 14.97 0 0.88 0 0.43 0 0.00 0 22.01 0 3199138 0 43 0 41.82 0 1.13 0 0.01 0 1.12 0 0.00 0 306.53 0 0.30 0 207891 0 4172273 0 624685 0 36725 0 17991 0 0 0 918419 0 17 0 0 0 508 0 85760 0 1348 0 87633 0 61.70 0 2574453 0 7662 99972 13.047768206735 4172273.0 3199138.0 207891.0 624685.0 36725.0 17991.0 0.0 918419.0 2574453.0 76.7 5.0 15.0 0.9 0.4 0.0 22.0 61.7 43 43 43.00 38 179407739 26.7 21.9 21.9 29.5 0.0 36.3 25.1 smartseq 1050905 SRR2088091 SRP060416 SRS980398 SRX1082060 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810526: T74_P1_F5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810526 GSM1810526 T74_P1_F5_ILC3 156640615 3642805 2016-01-28 01:00:06 172168137 156640615 3642805 1 3642805 index:0,count:3642805,average:43,stdev:0 GSM1810526_r1 6.43 7.54 0.22 107802658 137699450 86157882 113224578 127.73 131.42 0 0 0 0 0 0 59.27 75.31 4110744 1556491 4110744 1556491 66.25 72.57 4110744 1739643 4110744 1499831 15693805 14.56 6.06 0 15.35 0 0.95 0 0.45 0 0.00 0 26.51 0 2626046 0 43 0 41.69 0 1.15 0 0.01 0 1.15 0 0.00 0 84.06 0 0.34 0 220691 0 3642805 0 559280 0 34560 0 16551 0 0 0 965648 0 2 0 0 0 473 0 68593 0 1087 0 70155 0 56.74 0 2066766 0 5526 77957 14.107310893956 3642805.0 2626046.0 220691.0 559280.0 34560.0 16551.0 0.0 965648.0 2066766.0 72.1 6.1 15.4 0.9 0.5 0.0 26.5 56.7 43 43 43.00 38 156640615 26.4 21.8 21.9 29.9 0.0 35.6 24.0 smartseq 1050920 SRR2088092 SRP060416 SRS980396 SRX1082061 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810527: T74_P1_G1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810527 GSM1810527 T74_P1_G1_ILC3 225800224 5251168 2016-01-28 01:00:06 241842585 225800224 5251168 1 5251168 index:0,count:5251168,average:43,stdev:0 GSM1810527_r1 6.79 6.45 0.22 158391835 207426025 124849932 168882658 130.96 135.27 0 0 0 0 0 0 62.77 80.7 6222973 2412235 6222973 2412235 70.73 76.86 6222973 2718073 6222973 2297519 18153617 11.46 5.75 0 16.26 0 0.97 0 0.41 0 0.00 0 25.44 0 3842733 0 43 0 41.77 0 1.17 0 0.01 0 1.13 0 0.00 0 378.08 0 0.32 0 302190 0 5251168 0 853661 0 51178 0 21464 0 0 0 1335793 0 71 0 0 0 961 0 103228 0 1448 0 105708 0 56.92 0 2989072 0 6673 121720 18.240671362206 5251168.0 3842733.0 302190.0 853661.0 51178.0 21464.0 0.0 1335793.0 2989072.0 73.2 5.8 16.3 1.0 0.4 0.0 25.4 56.9 43 43 43.00 38 225800224 26.4 22.1 22.2 29.4 0.0 36.0 24.5 smartseq 1050937 SRR2088093 SRP060416 SRS980395 SRX1082062 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810528: T74_P1_G4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810528 GSM1810528 T74_P1_G4_NK 36469246 848122 2016-01-28 01:00:06 40092558 36469246 848122 1 848122 index:0,count:848122,average:43,stdev:0 GSM1810528_r1 5.63 5.81 0.29 27492530 36220996 22475202 30337040 131.75 134.98 0 0 0 0 0 0 65.31 80.66 993376 432659 993376 432659 71.49 76.22 993376 473567 993376 408841 2992799 10.89 4.64 0 14.86 0 0.92 0 0.44 0 0.00 0 20.53 0 662470 0 43 0 41.90 0 1.16 0 0.01 0 1.15 0 0.00 0 190.83 0 0.32 0 39312 0 848122 0 126058 0 7825 0 3736 0 0 0 174091 0 19 0 0 0 123 0 20678 0 262 0 21082 0 63.25 0 536412 0 6344 23842 3.758196721311 848122.0 662470.0 39312.0 126058.0 7825.0 3736.0 0.0 174091.0 536412.0 78.1 4.6 14.9 0.9 0.4 0.0 20.5 63.2 43 43 43.00 38 36469246 26.3 22.5 22.5 28.8 0.0 36.1 24.9 smartseq 1050954 SRR2088094 SRP060416 SRS980394 SRX1082063 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810529: T74_P1_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810529 GSM1810529 T74_P1_G9_NK 46908829 1090903 2016-01-28 01:00:06 52908356 46908829 1090903 1 1090903 index:0,count:1090903,average:43,stdev:0 GSM1810529_r1 4.38 7.81 0.32 33636643 42901119 27044801 35593177 127.54 131.61 0 0 0 0 0 0 59.78 75.5 1258421 490103 1258421 490103 65.7 72.05 1258421 538683 1258421 467686 4655251 13.84 5.27 0 15.65 0 0.98 0 0.42 0 0.00 0 23.44 0 819887 0 43 0 41.66 0 1.11 0 0.01 0 1.15 0 0.00 0 357.02 0 0.36 0 57465 0 1090903 0 170758 0 10708 0 4632 0 0 0 255676 0 2 0 0 0 119 0 21807 0 411 0 22339 0 59.50 0 649129 0 4032 25575 6.343005952381 1090903.0 819887.0 57465.0 170758.0 10708.0 4632.0 0.0 255676.0 649129.0 75.2 5.3 15.7 1.0 0.4 0.0 23.4 59.5 43 43 43.00 38 46908829 27.1 21.3 21.6 30.0 0.0 35.3 23.5 smartseq 1050970 SRR2088095 SRP060416 SRS980392 SRX1082064 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810530: T74_P1_H10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810530 GSM1810530 T74_P1_H10_ILC2 119269788 2773716 2016-01-28 01:00:06 129979872 119269788 2773716 1 2773716 index:0,count:2773716,average:43,stdev:0 GSM1810530_r1 5.44 6.36 0.24 90409597 119074913 74372176 100687112 131.71 135.38 0 0 0 0 0 0 66.7 82.06 3260450 1459653 3260450 1459653 72.15 78.07 3260450 1578971 3260450 1388571 9966548 11.02 4.50 0 14.77 0 0.86 0 0.41 0 0.00 0 19.83 0 2188453 0 43 0 41.81 0 1.12 0 0.01 0 1.15 0 0.00 0 312.04 0 0.31 0 124930 0 2773716 0 409796 0 23839 0 11311 0 0 0 550113 0 62 0 0 0 348 0 62433 0 816 0 63659 0 64.13 0 1778657 0 4995 72074 14.429229229229 2773716.0 2188453.0 124930.0 409796.0 23839.0 11311.0 0.0 550113.0 1778657.0 78.9 4.5 14.8 0.9 0.4 0.0 19.8 64.1 43 43 43.00 38 119269788 26.8 21.8 21.7 29.7 0.0 36.0 24.6 smartseq 1050986 SRR2088096 SRP060416 SRS979766 SRX1082065 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810531: T74_P2_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810531 GSM1810531 T74_P2_B4_NK 80214006 1865442 2016-01-28 01:00:06 87582913 80214006 1865442 1 1865442 index:0,count:1865442,average:43,stdev:0 GSM1810531_r1 4.16 6.87 0.2 51606338 64826048 40849376 53115187 125.62 130.03 0 0 0 0 0 0 57.68 74.08 2018273 725839 2018273 725839 64.46 71.06 2018273 811197 2018273 696280 8014447 15.53 7.05 0 14.94 0 1.05 0 0.49 0 0.00 0 31.00 0 1258486 0 43 0 41.69 0 1.18 0 0.01 0 1.16 0 0.00 0 248.73 0 0.32 0 131597 0 1865442 0 278668 0 19586 0 9109 0 0 0 578261 0 7 0 0 0 235 0 30608 0 512 0 31362 0 52.52 0 979818 0 4050 35492 8.763456790123 1865442.0 1258486.0 131597.0 278668.0 19586.0 9109.0 0.0 578261.0 979818.0 67.5 7.1 14.9 1.0 0.5 0.0 31.0 52.5 43 43 43.00 38 80214006 26.4 21.9 21.9 29.9 0.0 36.0 24.4 smartseq 1051003 SRR2088097 SRP060416 SRS980393 SRX1082066 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810532: T74_P2_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810532 GSM1810532 T74_P2_B9_NK 17302555 402385 2016-01-28 01:00:06 19061100 17302555 402385 1 402385 index:0,count:402385,average:43,stdev:0 GSM1810532_r1 7.0 5.08 0.24 14511913 19203563 12181118 16429461 132.33 134.88 0 0 0 0 0 0 66.76 80.11 503610 232830 503610 232830 72.36 76.57 503610 252361 503610 222544 1600518 11.03 2.68 0 14.45 0 0.89 0 0.55 0 0.00 0 11.89 0 348781 0 43 0 41.91 0 1.31 0 0.01 0 1.13 0 0.01 0 103.47 0 0.31 0 10772 0 402385 0 58151 0 3567 0 2194 0 0 0 47843 0 0 0 0 0 81 0 9444 0 120 0 9645 0 72.23 0 290630 0 3404 10799 3.172444183314 402385.0 348781.0 10772.0 58151.0 3567.0 2194.0 0.0 47843.0 290630.0 86.7 2.7 14.5 0.9 0.5 0.0 11.9 72.2 43 43 43.00 38 17302555 27.2 21.9 22.0 28.9 0.0 36.2 25.2 smartseq 1051018 SRR2088098 SRP060416 SRS980391 SRX1082067 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810533: T74_P2_C10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810533 GSM1810533 T74_P2_C10_ILC3 55331196 1286772 2016-01-28 01:00:06 60491615 55331196 1286772 1 1286772 index:0,count:1286772,average:43,stdev:0 GSM1810533_r1 7.02 5.52 0.17 43779426 57933363 35699476 48081865 132.33 134.69 0 0 0 0 0 0 66.9 82.68 1578041 704071 1578041 704071 74.06 78.38 1578041 779524 1578041 667495 4230264 9.66 3.82 0 15.61 0 0.86 0 0.51 0 0.00 0 16.84 0 1052490 0 43 0 41.92 0 1.18 0 0.01 0 1.13 0 0.00 0 330.88 0 0.31 0 49153 0 1286772 0 200880 0 11077 0 6576 0 0 0 216629 0 8 0 0 0 190 0 34327 0 387 0 34912 0 66.18 0 851610 0 6734 39844 5.916839916840 1286772.0 1052490.0 49153.0 200880.0 11077.0 6576.0 0.0 216629.0 851610.0 81.8 3.8 15.6 0.9 0.5 0.0 16.8 66.2 43 43 43.00 38 55331196 26.5 22.4 22.4 28.6 0.0 36.2 25.2 smartseq 1051034 SRR2088099 SRP060416 SRS980390 SRX1082068 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810534: T74_P2_C11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810534 GSM1810534 T74_P2_C11_ILC3 117624823 2735461 2016-01-28 01:00:06 128377391 117624823 2735461 1 2735461 index:0,count:2735461,average:43,stdev:0 GSM1810534_r1 6.0 7.07 0.28 80128510 100597033 63931137 82500108 125.54 129.05 0 0 0 0 0 0 58.08 73.96 3090503 1134422 3090503 1134422 64.25 70.79 3090503 1255014 3090503 1085829 12610053 15.74 6.11 0 15.33 0 1.03 0 0.54 0 0.00 0 27.02 0 1953275 0 43 0 41.68 0 1.20 0 0.01 0 1.19 0 0.00 0 246.19 0 0.32 0 167135 0 2735461 0 419427 0 28220 0 14722 0 0 0 739244 0 17 0 0 0 365 0 49215 0 967 0 50564 0 56.07 0 1533848 0 5041 55851 11.079349335449 2735461.0 1953275.0 167135.0 419427.0 28220.0 14722.0 0.0 739244.0 1533848.0 71.4 6.1 15.3 1.0 0.5 0.0 27.0 56.1 43 43 43.00 38 117624823 26.5 21.8 21.8 30.0 0.0 35.9 24.4 smartseq 1052681 SRR2088100 SRP060416 SRS980389 SRX1082069 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810535: T74_P2_C12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810535 GSM1810535 T74_P2_C12_ILC3 76874712 1787784 2016-01-28 01:00:06 84288901 76874712 1787784 1 1787784 index:0,count:1787784,average:43,stdev:0 GSM1810535_r1 4.54 7.61 0.25 51814248 63542448 41831055 52995440 122.64 126.69 0 0 0 0 0 0 55.53 70.03 1998732 703553 1998732 703553 60.82 67.33 1998732 770643 1998732 676446 9766166 18.85 6.26 0 14.68 0 1.05 0 0.42 0 0.00 0 27.66 0 1267053 0 43 0 41.64 0 1.24 0 0.01 0 1.10 0 0.00 0 68.47 0 0.32 0 111895 0 1787784 0 262369 0 18715 0 7484 0 0 0 494532 0 6 0 0 0 222 0 26288 0 632 0 27148 0 56.20 0 1004684 0 3665 30452 8.308867667121 1787784.0 1267053.0 111895.0 262369.0 18715.0 7484.0 0.0 494532.0 1004684.0 70.9 6.3 14.7 1.0 0.4 0.0 27.7 56.2 43 43 43.00 38 76874712 27.0 21.3 21.3 30.4 0.0 35.9 24.1 smartseq 1052698 SRR2088101 SRP060416 SRS980387 SRX1082070 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810536: T74_P2_C2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810536 GSM1810536 T74_P2_C2_ILC3 63152208 1468656 2016-01-28 01:00:06 69208696 63152208 1468656 1 1468656 index:0,count:1468656,average:43,stdev:0 GSM1810536_r1 6.36 5.71 0.24 45652327 58263930 37205285 48728079 127.63 130.97 0 0 0 0 0 0 59.36 73.64 1690253 653606 1690253 653606 64.87 69.58 1690253 714320 1690253 617581 6607121 14.47 5.24 0 14.54 0 1.23 0 0.53 0 0.00 0 23.26 0 1101091 0 43 0 41.92 0 1.16 0 0.01 0 1.14 0 0.00 0 293.73 0 0.32 0 77001 0 1468656 0 213514 0 18109 0 7843 0 0 0 341613 0 3 0 0 0 214 0 28302 0 414 0 28933 0 60.43 0 887577 0 5315 32071 6.034054562559 1468656.0 1101091.0 77001.0 213514.0 18109.0 7843.0 0.0 341613.0 887577.0 75.0 5.2 14.5 1.2 0.5 0.0 23.3 60.4 43 43 43.00 38 63152208 26.3 22.4 22.4 28.9 0.0 36.1 24.8 smartseq 1052714 SRR2088102 SRP060416 SRS980388 SRX1082071 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810537: T74_P2_C3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810537 GSM1810537 T74_P2_C3_ILC3 146063604 3396828 2016-01-28 01:00:06 158872146 146063604 3396828 1 3396828 index:0,count:3396828,average:43,stdev:0 GSM1810537_r1 10.96 5.25 0.25 111237769 150796608 90331872 125047361 135.56 138.43 0 0 0 0 0 0 63.94 79.47 4046200 1708651 4046200 1708651 71.43 75.78 4046200 1908819 4046200 1629236 12579318 11.31 4.53 0 15.37 0 0.78 0 0.51 0 0.00 0 20.05 0 2672132 0 43 0 42.02 0 1.12 0 0.01 0 1.12 0 0.00 0 76.43 0 0.30 0 154046 0 3396828 0 522162 0 26433 0 17191 0 0 0 681072 0 35 0 0 0 496 0 75302 0 1032 0 76865 0 63.29 0 2149970 0 8801 86528 9.831610044313 3396828.0 2672132.0 154046.0 522162.0 26433.0 17191.0 0.0 681072.0 2149970.0 78.7 4.5 15.4 0.8 0.5 0.0 20.1 63.3 43 43 43.00 38 146063604 26.5 22.3 22.3 28.9 0.0 36.0 25.0 smartseq 1052730 SRR2088103 SRP060416 SRS980386 SRX1082072 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810538: T74_P2_C4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810538 GSM1810538 T74_P2_C4_ILC3 131035018 3047326 2016-01-28 01:00:06 141733661 131035018 3047326 1 3047326 index:0,count:3047326,average:43,stdev:0 GSM1810538_r1 8.77 6.3 0.18 101093134 137860716 83286850 115576967 136.37 138.77 0 0 0 0 0 0 66.99 82.06 3519366 1628192 3519366 1628192 74.07 78.32 3519366 1800234 3519366 1554132 10126059 10.02 4.31 0 14.64 0 0.75 0 0.52 0 0.00 0 18.97 0 2430492 0 43 0 41.97 0 1.14 0 0.01 0 1.12 0 0.00 0 288.69 0 0.31 0 131243 0 3047326 0 446237 0 22855 0 15834 0 0 0 578145 0 16 0 0 0 377 0 74695 0 786 0 75874 0 65.11 0 1984255 0 7701 85651 11.122062069861 3047326.0 2430492.0 131243.0 446237.0 22855.0 15834.0 0.0 578145.0 1984255.0 79.8 4.3 14.6 0.8 0.5 0.0 19.0 65.1 43 43 43.00 38 131035018 26.6 22.2 22.3 28.8 0.0 36.2 25.0 smartseq 1052748 SRR2088104 SRP060416 SRS980385 SRX1082073 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810539: T74_P2_C5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810539 GSM1810539 T74_P2_C5_ILC3 143250415 3331405 2016-01-28 01:00:06 154184630 143250415 3331405 1 3331405 index:0,count:3331405,average:43,stdev:0 GSM1810539_r1 10.91 5.2 0.17 114666584 157464389 92562906 129009653 137.32 139.38 0 0 0 0 0 0 65.3 81.5 4144331 1795896 4144331 1795896 74.62 77.81 4144331 2052298 4144331 1714638 11833165 10.32 3.68 0 16.41 0 0.87 0 0.44 0 0.00 0 16.13 0 2750346 0 43 0 42.01 0 1.10 0 0.01 0 1.15 0 0.00 0 307.51 0 0.31 0 122650 0 3331405 0 546835 0 29047 0 14737 0 0 0 537275 0 17 0 0 0 467 0 80619 0 869 0 81972 0 66.14 0 2203511 0 9708 95621 9.849711578080 3331405.0 2750346.0 122650.0 546835.0 29047.0 14737.0 0.0 537275.0 2203511.0 82.6 3.7 16.4 0.9 0.4 0.0 16.1 66.1 43 43 43.00 38 143250415 26.6 22.4 22.5 28.5 0.0 36.3 25.3 smartseq 1052764 SRR2088105 SRP060416 SRS980384 SRX1082074 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810540: T74_P2_C6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810540 GSM1810540 T74_P2_C6_ILC3 150386222 3497354 2016-01-28 01:00:06 164202693 150386222 3497354 1 3497354 index:0,count:3497354,average:43,stdev:0 GSM1810540_r1 8.49 5.5 0.2 113718871 152655248 92099011 126613884 134.24 137.48 0 0 0 0 0 0 64.31 80.2 4115726 1761933 4115726 1761933 71.46 75.72 4115726 1957571 4115726 1663563 12945196 11.38 4.59 0 15.51 0 0.89 0 0.42 0 0.00 0 20.36 0 2739569 0 43 0 41.92 0 1.15 0 0.01 0 1.15 0 0.00 0 299.77 0 0.33 0 160418 0 3497354 0 542611 0 31006 0 14582 0 0 0 712197 0 32 0 0 0 498 0 86924 0 931 0 88385 0 62.82 0 2196958 0 9206 101684 11.045405170541 3497354.0 2739569.0 160418.0 542611.0 31006.0 14582.0 0.0 712197.0 2196958.0 78.3 4.6 15.5 0.9 0.4 0.0 20.4 62.8 43 43 43.00 38 150386222 26.3 22.4 22.5 28.8 0.0 35.9 24.7 smartseq 1052777 SRR2088106 SRP060416 SRS980383 SRX1082075 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810541: T74_P2_C8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810541 GSM1810541 T74_P2_C8_ILC3 69831226 1623982 2016-01-28 01:00:06 78219304 69831226 1623982 1 1623982 index:0,count:1623982,average:43,stdev:0 GSM1810541_r1 3.87 9.13 0.27 43225170 51852728 33794828 42088897 119.96 124.54 0 0 0 0 0 0 55.22 72.31 1766574 590334 1766574 590334 60.65 69.5 1766574 648330 1766574 567404 7687692 17.79 7.39 0 15.55 0 1.07 0 0.35 0 0.00 0 32.75 0 1068990 0 43 0 41.39 0 1.27 0 0.01 0 1.19 0 0.00 0 167.04 0 0.37 0 120089 0 1623982 0 252561 0 17364 0 5763 0 0 0 531865 0 3 0 0 0 148 0 21335 0 433 0 21919 0 50.27 0 816429 0 2765 23934 8.656057866184 1623982.0 1068990.0 120089.0 252561.0 17364.0 5763.0 0.0 531865.0 816429.0 65.8 7.4 15.6 1.1 0.4 0.0 32.8 50.3 43 43 43.00 38 69831226 26.8 20.8 21.1 31.3 0.0 35.2 23.2 smartseq 1052793 SRR2088107 SRP060416 SRS980381 SRX1082076 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810542: T74_P2_C9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810542 GSM1810542 T74_P2_C9_ILC3 56393898 1311486 2016-01-28 01:00:06 62325718 56393898 1311486 1 1311486 index:0,count:1311486,average:43,stdev:0 GSM1810542_r1 4.83 5.6 0.18 44626886 57827790 38071078 50261329 129.58 132.02 0 0 0 0 0 0 67.86 80.26 1505052 729172 1505052 729172 72.69 77.1 1505052 781073 1505052 700491 4976373 11.15 3.80 0 12.66 0 0.82 0 0.48 0 0.00 0 16.77 0 1074584 0 43 0 41.91 0 1.25 0 0.01 0 1.15 0 0.00 0 337.24 0 0.32 0 49801 0 1311486 0 166076 0 10697 0 6332 0 0 0 219873 0 15 0 0 0 248 0 28069 0 356 0 28688 0 69.27 0 908508 0 3958 32159 8.125063163214 1311486.0 1074584.0 49801.0 166076.0 10697.0 6332.0 0.0 219873.0 908508.0 81.9 3.8 12.7 0.8 0.5 0.0 16.8 69.3 43 43 43.00 38 56393898 27.1 21.9 22.2 28.8 0.0 35.9 24.6 smartseq 1052809 SRR2088108 SRP060416 SRS980382 SRX1082077 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810543: T74_P2_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810543 GSM1810543 T74_P2_D11_NK 89543071 2082397 2016-01-28 01:00:06 97507077 89543071 2082397 1 2082397 index:0,count:2082397,average:43,stdev:0 GSM1810543_r1 4.44 6.68 0.28 62434550 79973402 50300187 66402240 128.09 132.01 0 0 0 0 0 0 62.4 78.57 2359411 946163 2359411 946163 68.59 74.97 2359411 1040025 2359411 902875 7917768 12.68 5.87 0 14.98 0 0.93 0 0.43 0 0.00 0 25.82 0 1516298 0 43 0 41.77 0 1.25 0 0.01 0 1.13 0 0.00 0 81.49 0 0.33 0 122250 0 2082397 0 312026 0 19365 0 9037 0 0 0 537697 0 58 0 0 0 269 0 43242 0 718 0 44287 0 57.83 0 1204272 0 5742 49834 8.678857540927 2082397.0 1516298.0 122250.0 312026.0 19365.0 9037.0 0.0 537697.0 1204272.0 72.8 5.9 15.0 0.9 0.4 0.0 25.8 57.8 43 43 43.00 38 89543071 26.3 22.0 22.1 29.5 0.0 36.0 24.5 smartseq 1052825 SRR2088109 SRP060416 SRS980380 SRX1082078 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810544: T74_P2_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810544 GSM1810544 T74_P2_D2_NK 65481518 1522826 2016-01-28 01:00:06 73843125 65481518 1522826 1 1522826 index:0,count:1522826,average:43,stdev:0 GSM1810544_r1 9.95 6.62 0.23 46684535 61858851 37028636 50504214 132.5 136.39 0 0 0 0 0 0 62.3 79.65 1788199 705575 1788199 705575 70.03 76.24 1788199 793144 1788199 675294 5631789 12.06 5.54 0 16.21 0 0.90 0 0.32 0 0.00 0 24.40 0 1132610 0 43 0 41.80 0 1.11 0 0.01 0 1.13 0 0.00 0 219.29 0 0.36 0 84298 0 1522826 0 246821 0 13744 0 4859 0 0 0 371613 0 14 0 0 0 221 0 29528 0 408 0 30171 0 58.17 0 885789 0 5985 34498 5.764076858814 1522826.0 1132610.0 84298.0 246821.0 13744.0 4859.0 0.0 371613.0 885789.0 74.4 5.5 16.2 0.9 0.3 0.0 24.4 58.2 43 43 43.00 38 65481518 26.6 21.7 22.0 29.7 0.0 35.2 23.6 smartseq 1052939 SRR2088110 SRP060416 SRS980379 SRX1082079 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810545: T74_P2_E12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810545 GSM1810545 T74_P2_E12_ILC3 87579519 2036733 2016-01-28 01:00:06 95339667 87579519 2036733 1 2036733 index:0,count:2036733,average:43,stdev:0 GSM1810545_r1 5.29 6.81 0.17 59913807 78786755 46952366 64162759 131.5 136.66 0 0 0 0 0 0 62.17 80.5 2385186 906335 2385186 906335 69.95 76.86 2385186 1019801 2385186 865308 6944854 11.59 6.14 0 16.30 0 1.07 0 0.34 0 0.00 0 27.01 0 1457824 0 43 0 41.70 0 1.14 0 0.01 0 1.15 0 0.00 0 222.19 0 0.33 0 125069 0 2036733 0 331932 0 21784 0 6988 0 0 0 550137 0 5 0 0 0 294 0 41020 0 617 0 41936 0 55.28 0 1125892 0 5485 49479 9.020783956244 2036733.0 1457824.0 125069.0 331932.0 21784.0 6988.0 0.0 550137.0 1125892.0 71.6 6.1 16.3 1.1 0.3 0.0 27.0 55.3 43 43 43.00 38 87579519 26.4 22.0 22.0 29.6 0.0 36.0 24.4 smartseq 1052952 SRR2088111 SRP060416 SRS980377 SRX1082080 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810546: T74_P2_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810546 GSM1810546 T74_P2_E7_NK 48091114 1118398 2016-01-28 01:00:06 53365277 48091114 1118398 1 1118398 index:0,count:1118398,average:43,stdev:0 GSM1810546_r1 4.42 7.54 0.32 32892566 40476639 26017925 33037189 123.06 126.98 0 0 0 0 0 0 60.21 77.35 1276657 484247 1276657 484247 66.32 73.84 1276657 533413 1276657 462296 4644829 14.12 6.02 0 15.94 0 1.13 0 0.43 0 0.00 0 26.52 0 804313 0 43 0 41.56 0 1.24 0 0.01 0 1.08 0 0.00 0 201.31 0 0.34 0 67295 0 1118398 0 178273 0 12646 0 4816 0 0 0 296623 0 15 0 0 0 111 0 20258 0 355 0 20739 0 55.98 0 626040 0 3827 23321 6.093807159655 1118398.0 804313.0 67295.0 178273.0 12646.0 4816.0 0.0 296623.0 626040.0 71.9 6.0 15.9 1.1 0.4 0.0 26.5 56.0 43 43 43.00 38 48091114 26.9 21.4 21.4 30.3 0.0 35.7 24.0 smartseq 1052969 SRR2088112 SRP060416 SRS980376 SRX1082081 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810547: T74_P2_F1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810547 GSM1810547 T74_P2_F1_ILC3 135520090 3151630 2016-01-28 01:00:06 147684799 135520090 3151630 1 3151630 index:0,count:3151630,average:43,stdev:0 GSM1810547_r1 9.22 5.97 0.22 104536330 140123229 86044166 117461896 134.04 136.51 0 0 0 0 0 0 64.53 79.13 3731145 1622344 3731145 1622344 71.53 75.37 3731145 1798245 3731145 1545285 12105110 11.58 4.31 0 14.71 0 0.83 0 0.41 0 0.00 0 18.99 0 2513980 0 43 0 41.97 0 1.17 0 0.01 0 1.21 0 0.00 0 366.00 0 0.32 0 135792 0 3151630 0 463632 0 26116 0 12923 0 0 0 598611 0 9 0 0 0 519 0 76790 0 953 0 78271 0 65.06 0 2050348 0 8984 87481 9.737422083704 3151630.0 2513980.0 135792.0 463632.0 26116.0 12923.0 0.0 598611.0 2050348.0 79.8 4.3 14.7 0.8 0.4 0.0 19.0 65.1 43 43 43.00 38 135520090 26.7 22.2 22.3 28.8 0.0 36.0 24.8 smartseq 1052985 SRR2088113 SRP060416 SRS980378 SRX1082082 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810548: T74_P2_G4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810548 GSM1810548 T74_P2_G4_NK 77283169 1797283 2016-01-28 01:00:06 86588405 77283169 1797283 1 1797283 index:0,count:1797283,average:43,stdev:0 GSM1810548_r1 9.28 5.92 0.28 61567612 83788652 50401899 69757045 136.09 138.4 0 0 0 0 0 0 66.54 81.96 2176939 982952 2176939 982952 74.17 78.11 2176939 1095682 2176939 936829 6130446 9.96 3.75 0 15.47 0 0.75 0 0.45 0 0.00 0 16.60 0 1477326 0 43 0 42.02 0 1.14 0 0.01 0 1.13 0 0.00 0 308.11 0 0.36 0 67349 0 1797283 0 277965 0 13555 0 8133 0 0 0 298269 0 28 0 0 0 241 0 44647 0 451 0 45367 0 66.73 0 1199361 0 7574 52847 6.977422762081 1797283.0 1477326.0 67349.0 277965.0 13555.0 8133.0 0.0 298269.0 1199361.0 82.2 3.7 15.5 0.8 0.5 0.0 16.6 66.7 43 43 43.00 38 77283169 26.9 22.0 22.5 28.6 0.0 35.4 24.1 smartseq 1053001 SRR2088114 SRP060416 SRS980375 SRX1082083 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810549: T74_P2_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810549 GSM1810549 T74_P2_G9_NK 107405572 2497804 2016-01-28 01:00:06 116558905 107405572 2497804 1 2497804 index:0,count:2497804,average:43,stdev:0 GSM1810549_r1 7.8 5.03 0.17 87213453 117322426 70988497 97281719 134.52 137.04 0 0 0 0 0 0 64.69 80.1 3180431 1356213 3180431 1356213 72.52 76.2 3180431 1520288 3180431 1290146 8776205 10.06 3.27 0 16.15 0 1.08 0 0.57 0 0.00 0 14.42 0 2096486 0 43 0 41.93 0 1.17 0 0.01 0 1.14 0 0.00 0 408.73 0 0.31 0 81576 0 2497804 0 403379 0 26907 0 14320 0 0 0 360091 0 0 0 0 0 326 0 55041 0 743 0 56110 0 67.78 0 1693107 0 5880 66079 11.237925170068 2497804.0 2096486.0 81576.0 403379.0 26907.0 14320.0 0.0 360091.0 1693107.0 83.9 3.3 16.1 1.1 0.6 0.0 14.4 67.8 43 43 43.00 38 107405572 27.0 22.1 22.2 28.7 0.0 36.2 25.1 smartseq 1053017 SRR2088115 SRP060416 SRS980374 SRX1082084 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810550: T74_P3_A11_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810550 GSM1810550 T74_P3_A11_ILC2 140675016 3271512 2016-01-28 01:00:06 151914756 140675016 3271512 1 3271512 index:0,count:3271512,average:43,stdev:0 GSM1810550_r1 2.08 5.16 0.18 98375216 120837175 75795972 99691480 122.83 131.53 0 0 0 0 0 0 58.83 77.29 4521226 1400583 4521226 1400583 64.81 73.49 4521226 1543002 4521226 1331771 12412742 12.62 5.25 0 17.38 0 0.94 0 0.35 0 0.00 0 25.94 0 2380635 0 43 0 41.83 0 1.34 0 0.00 0 1.14 0 0.00 0 267.67 0 0.31 0 171708 0 3271512 0 568455 0 30678 0 11605 0 0 0 848594 0 37 0 0 0 725 0 67158 0 873 0 68793 0 55.39 0 1812180 0 7710 78881 10.230998702983 3271512.0 2380635.0 171708.0 568455.0 30678.0 11605.0 0.0 848594.0 1812180.0 72.8 5.2 17.4 0.9 0.4 0.0 25.9 55.4 43 43 43.00 38 140675016 26.2 22.4 22.3 29.1 0.0 36.1 24.6 smartseq 1053032 SRR2088116 SRP060416 SRS980373 SRX1082085 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810551: T74_P3_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810551 GSM1810551 T74_P3_B4_NK 108428198 2521586 2016-01-28 01:00:06 120113815 108428198 2521586 1 2521586 index:0,count:2521586,average:43,stdev:0 GSM1810551_r1 3.21 4.1 0.18 82191941 102415271 63001231 84302118 124.61 133.81 0 0 0 0 0 0 58.67 77.17 3848227 1158194 3848227 1158194 64.86 72.62 3848227 1280390 3848227 1089947 9528327 11.59 4.21 0 18.77 0 0.93 0 0.44 0 0.00 0 20.35 0 1974033 0 43 0 41.98 0 1.35 0 0.00 0 1.12 0 0.00 0 324.20 0 0.34 0 106192 0 2521586 0 473191 0 23368 0 11056 0 0 0 513129 0 9 0 0 0 503 0 61707 0 687 0 62906 0 59.52 0 1500842 0 9371 71496 7.629495251307 2521586.0 1974033.0 106192.0 473191.0 23368.0 11056.0 0.0 513129.0 1500842.0 78.3 4.2 18.8 0.9 0.4 0.0 20.3 59.5 43 43 43.00 38 108428198 26.1 22.6 22.8 28.5 0.0 35.7 24.4 smartseq 1053050 SRR2088117 SRP060416 SRS980371 SRX1082086 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810552: T74_P3_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810552 GSM1810552 T74_P3_B9_NK 41900662 974434 2016-01-28 01:00:06 47241786 41900662 974434 1 974434 index:0,count:974434,average:43,stdev:0 GSM1810552_r1 1.36 5.36 0.27 29910843 36541888 23661325 30475802 122.17 128.8 0 0 0 0 0 0 58.4 74.74 1286119 422396 1286119 422396 63.99 71.02 1286119 462820 1286119 401370 4182389 13.98 5.04 0 16.23 0 0.99 0 0.40 0 0.00 0 24.38 0 723312 0 43 0 41.87 0 1.63 0 0.00 0 1.19 0 0.00 0 250.57 0 0.36 0 49136 0 974434 0 158144 0 9665 0 3890 0 0 0 237567 0 10 0 0 0 213 0 20611 0 260 0 21094 0 58.00 0 565168 0 5542 24260 4.377481053771 974434.0 723312.0 49136.0 158144.0 9665.0 3890.0 0.0 237567.0 565168.0 74.2 5.0 16.2 1.0 0.4 0.0 24.4 58.0 43 43 43.00 38 41900662 26.7 22.0 22.3 29.1 0.0 35.3 23.8 smartseq 1053068 SRR2088118 SRP060416 SRS980372 SRX1082087 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810553: T74_P3_C2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810553 GSM1810553 T74_P3_C2_ILC2 42976565 999455 2016-01-28 01:00:06 47207348 42976565 999455 1 999455 index:0,count:999455,average:43,stdev:0 GSM1810553_r1 1.75 5.62 0.19 28889064 35430599 22183625 29036945 122.64 130.89 0 0 0 0 0 0 58.78 77.55 1314235 411539 1314235 411539 64.98 73.77 1314235 454988 1314235 391492 3499710 12.11 5.82 0 16.96 0 0.94 0 0.32 0 0.00 0 28.69 0 700170 0 43 0 41.80 0 1.38 0 0.00 0 1.25 0 0.00 0 211.65 0 0.31 0 58140 0 999455 0 169465 0 9417 0 3158 0 0 0 286710 0 18 0 0 0 153 0 20536 0 293 0 21000 0 53.10 0 530705 0 5068 23699 4.676203630624 999455.0 700170.0 58140.0 169465.0 9417.0 3158.0 0.0 286710.0 530705.0 70.1 5.8 17.0 0.9 0.3 0.0 28.7 53.1 43 43 43.00 38 42976565 25.9 22.4 22.2 29.5 0.0 36.0 24.6 smartseq 1053083 SRR2088119 SRP060416 SRS980369 SRX1082088 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810554: T74_P3_C6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810554 GSM1810554 T74_P3_C6_ILC2 122090459 2839313 2016-01-28 01:00:06 132232619 122090459 2839313 1 2839313 index:0,count:2839313,average:43,stdev:0 GSM1810554_r1 2.16 4.62 0.15 92563821 115560254 73940831 97216427 124.84 131.48 0 0 0 0 0 0 60.52 76.43 3872900 1347026 3872900 1347026 65.88 71.94 3872900 1466274 3872900 1267993 11319465 12.23 4.12 0 16.31 0 1.00 0 0.49 0 0.00 0 20.12 0 2225637 0 43 0 41.95 0 1.40 0 0.00 0 1.15 0 0.00 0 425.90 0 0.32 0 116916 0 2839313 0 463175 0 28375 0 13990 0 0 0 571311 0 31 0 0 0 573 0 72414 0 851 0 73869 0 62.07 0 1762462 0 9983 84351 8.449464088951 2839313.0 2225637.0 116916.0 463175.0 28375.0 13990.0 0.0 571311.0 1762462.0 78.4 4.1 16.3 1.0 0.5 0.0 20.1 62.1 43 43 43.00 38 122090459 26.2 22.6 22.7 28.5 0.0 36.1 24.9 smartseq 1053195 SRR2088120 SRP060416 SRS980370 SRX1082089 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810555: T74_P3_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810555 GSM1810555 T74_P3_D11_NK 105535717 2454319 2016-01-28 01:00:06 114708911 105535717 2454319 1 2454319 index:0,count:2454319,average:43,stdev:0 GSM1810555_r1 4.19 3.95 0.23 88964389 115319618 69055588 93848737 129.62 135.9 0 0 0 0 0 0 61.99 80.22 3793402 1315098 3793402 1315098 70.44 75.48 3793402 1494351 3793402 1237391 8636787 9.71 2.55 0 19.65 0 0.97 0 0.44 0 0.00 0 12.15 0 2121520 0 43 0 42.12 0 1.34 0 0.00 0 1.15 0 0.00 0 384.15 0 0.30 0 62688 0 2454319 0 482195 0 23779 0 10875 0 0 0 298145 0 32 0 0 0 562 0 75403 0 650 0 76647 0 66.79 0 1639325 0 14717 90529 6.151321600870 2454319.0 2121520.0 62688.0 482195.0 23779.0 10875.0 0.0 298145.0 1639325.0 86.4 2.6 19.6 1.0 0.4 0.0 12.1 66.8 43 43 43.00 38 105535717 26.4 22.9 23.1 27.7 0.0 36.2 25.3 smartseq 1053211 SRR2088121 SRP060416 SRS980368 SRX1082090 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810556: T74_P3_D6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810556 GSM1810556 T74_P3_D6_ILC3 29140111 677677 2016-01-28 01:00:06 31867805 29140111 677677 1 677677 index:0,count:677677,average:43,stdev:0 GSM1810556_r1 4.03 5.59 0.21 20364779 25181054 16384616 21229852 123.65 129.57 0 0 0 0 0 0 56.87 71.52 830167 279264 830167 279264 61.51 67.67 830167 302066 830167 264246 2993790 14.70 5.32 0 14.84 0 0.88 0 0.47 0 0.00 0 26.19 0 491045 0 43 0 41.96 0 1.41 0 0.00 0 1.18 0 0.00 0 162.64 0 0.30 0 36084 0 677677 0 100548 0 5951 0 3172 0 0 0 177509 0 5 0 0 0 95 0 13469 0 192 0 13761 0 57.62 0 390497 0 5004 15061 3.009792166267 677677.0 491045.0 36084.0 100548.0 5951.0 3172.0 0.0 177509.0 390497.0 72.5 5.3 14.8 0.9 0.5 0.0 26.2 57.6 43 43 43.00 38 29140111 26.5 22.2 22.1 29.2 0.0 36.2 25.0 smartseq 1053227 SRR2088122 SRP060416 SRS980366 SRX1082091 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810557: T74_P3_D7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810557 GSM1810557 T74_P3_D7_ILC3 23550928 547696 2016-01-28 01:00:06 25779645 23550928 547696 1 547696 index:0,count:547696,average:43,stdev:0 GSM1810557_r1 2.91 6.54 0.27 15581958 18934042 12167053 15518905 121.51 127.55 0 0 0 0 0 0 59.22 76.92 663618 224026 663618 224026 64.92 72.68 663618 245586 663618 211677 2043634 13.12 5.93 0 15.89 0 0.90 0 0.40 0 0.00 0 29.64 0 378299 0 43 0 41.78 0 1.36 0 0.00 0 1.15 0 0.00 0 164.31 0 0.31 0 32492 0 547696 0 87048 0 4904 0 2183 0 0 0 162310 0 1 0 0 0 130 0 11594 0 154 0 11879 0 53.18 0 291251 0 4481 13052 2.912742691364 547696.0 378299.0 32492.0 87048.0 4904.0 2183.0 0.0 162310.0 291251.0 69.1 5.9 15.9 0.9 0.4 0.0 29.6 53.2 43 43 43.00 38 23550928 26.3 22.1 22.0 29.5 0.0 36.2 24.8 smartseq 1053243 SRR2088123 SRP060416 SRS980367 SRX1082092 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810558: T74_P3_E4_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810558 GSM1810558 T74_P3_E4_ILC2 127880409 2973963 2016-01-28 01:00:06 138317269 127880409 2973963 1 2973963 index:0,count:2973963,average:43,stdev:0 GSM1810558_r1 1.84 5.3 0.19 93558444 115699715 73313988 96249804 123.67 131.28 0 0 0 0 0 0 59.28 76.39 4093495 1335728 4093495 1335728 66.06 72.9 4093495 1488572 4093495 1274665 11172913 11.94 4.61 0 16.97 0 0.89 0 0.44 0 0.00 0 22.91 0 2253251 0 43 0 41.93 0 1.34 0 0.00 0 1.14 0 0.00 0 65.28 0 0.31 0 137075 0 2973963 0 504693 0 26453 0 13067 0 0 0 681192 0 4 0 0 0 540 0 70699 0 783 0 72026 0 58.80 0 1748558 0 9189 84467 9.192186309718 2973963.0 2253251.0 137075.0 504693.0 26453.0 13067.0 0.0 681192.0 1748558.0 75.8 4.6 17.0 0.9 0.4 0.0 22.9 58.8 43 43 43.00 38 127880409 26.2 22.5 22.5 28.8 0.0 36.1 24.9 smartseq 1053259 SRR2088124 SRP060416 SRS980365 SRX1082093 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810559: T74_P3_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810559 GSM1810559 T74_P3_E7_NK 88417460 2056220 2016-01-28 01:00:06 96937851 88417460 2056220 1 2056220 index:0,count:2056220,average:43,stdev:0 GSM1810559_r1 2.78 6.84 0.24 56169172 68453433 44107188 56576705 121.87 128.27 0 0 0 0 0 0 53.63 69.47 2345814 735070 2345814 735070 59.03 66.41 2345814 809065 2345814 702716 9944848 17.71 6.42 0 15.20 0 1.00 0 0.47 0 0.00 0 31.87 0 1370651 0 43 0 41.68 0 1.34 0 0.00 0 1.16 0 0.00 0 255.25 0 0.32 0 132017 0 2056220 0 312539 0 20643 0 9614 0 0 0 655312 0 4 0 0 0 456 0 32960 0 536 0 33956 0 51.46 0 1058112 0 4886 39470 8.078182562423 2056220.0 1370651.0 132017.0 312539.0 20643.0 9614.0 0.0 655312.0 1058112.0 66.7 6.4 15.2 1.0 0.5 0.0 31.9 51.5 43 43 43.00 38 88417460 26.2 22.0 21.9 29.9 0.0 35.9 24.3 smartseq 1053275 SRR2088125 SRP060416 SRS980364 SRX1082094 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810560: T74_P3_E9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810560 GSM1810560 T74_P3_E9_ILC2 122769171 2855097 2016-01-28 01:00:06 134037717 122769171 2855097 1 2855097 index:0,count:2855097,average:43,stdev:0 GSM1810560_r1 2.33 6.88 0.21 78498338 95107277 60930397 78422384 121.16 128.71 0 0 0 0 0 0 57.21 74.93 3447426 1095812 3447426 1095812 63.15 71.7 3447426 1209631 3447426 1048595 11915705 15.18 6.41 0 15.87 0 0.91 0 0.30 0 0.00 0 31.70 0 1915423 0 43 0 41.66 0 1.44 0 0.00 0 1.14 0 0.00 0 342.61 0 0.32 0 183102 0 2855097 0 452965 0 25907 0 8571 0 0 0 905196 0 45 0 0 0 407 0 50285 0 816 0 51553 0 51.22 0 1462458 0 5768 59687 10.347954230236 2855097.0 1915423.0 183102.0 452965.0 25907.0 8571.0 0.0 905196.0 1462458.0 67.1 6.4 15.9 0.9 0.3 0.0 31.7 51.2 43 43 43.00 38 122769171 26.2 22.0 21.8 30.0 0.0 35.9 24.4 smartseq 1053292 SRR2088126 SRP060416 SRS980363 SRX1082095 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810561: T74_P3_F1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810561 GSM1810561 T74_P3_F1_ILC3 27507358 639706 2016-01-28 01:00:06 30322005 27507358 639706 1 639706 index:0,count:639706,average:43,stdev:0 GSM1810561_r1 3.15 5.63 0.28 17874474 21025159 13714967 17113745 117.63 124.78 0 0 0 0 0 0 51.63 68.32 822030 224055 822030 224055 57.5 65.24 822030 249517 822030 213967 3032228 16.96 6.29 0 16.56 0 1.01 0 0.48 0 0.00 0 30.68 0 433921 0 43 0 41.82 0 1.50 0 0.00 0 1.17 0 0.01 0 164.50 0 0.31 0 40229 0 639706 0 105958 0 6469 0 3070 0 0 0 196246 0 3 0 0 0 102 0 8028 0 215 0 8348 0 51.27 0 327963 0 2969 9422 3.173459077130 639706.0 433921.0 40229.0 105958.0 6469.0 3070.0 0.0 196246.0 327963.0 67.8 6.3 16.6 1.0 0.5 0.0 30.7 51.3 43 43 43.00 38 27507358 26.5 21.9 21.7 29.8 0.0 36.0 24.6 smartseq 1053308 SRR2088127 SRP060416 SRS980362 SRX1082096 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810562: T74_P3_F2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810562 GSM1810562 T74_P3_F2_ILC3 23711533 551431 2016-01-28 01:00:06 25994847 23711533 551431 1 551431 index:0,count:551431,average:43,stdev:0 GSM1810562_r1 2.07 6.18 0.19 14754255 17557832 11362700 14331364 119.0 126.13 0 0 0 0 0 0 55.85 73.7 662627 200625 662627 200625 61.53 69.97 662627 221051 662627 190465 2382035 16.14 6.75 0 15.78 0 0.89 0 0.39 0 0.00 0 33.57 0 359235 0 43 0 41.74 0 1.43 0 0.00 0 1.12 0 0.00 0 116.77 0 0.32 0 37226 0 551431 0 87021 0 4897 0 2177 0 0 0 185122 0 3 0 0 0 100 0 9411 0 180 0 9694 0 49.37 0 272214 0 3980 11192 2.812060301508 551431.0 359235.0 37226.0 87021.0 4897.0 2177.0 0.0 185122.0 272214.0 65.1 6.8 15.8 0.9 0.4 0.0 33.6 49.4 43 43 43.00 38 23711533 26.3 22.0 21.9 29.9 0.0 36.1 24.6 smartseq 1053323 SRR2088128 SRP060416 SRS979767 SRX1082097 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810563: T74_P3_F3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810563 GSM1810563 T74_P3_F3_ILC3 27389108 636956 2016-01-28 01:00:06 29906928 27389108 636956 1 636956 index:0,count:636956,average:43,stdev:0 GSM1810563_r1 1.33 5.71 0.22 17860790 21123906 14068208 17658290 118.27 125.52 0 0 0 0 0 0 52.56 67.69 781543 227578 781543 227578 57.48 64.43 781543 248913 781543 216622 3157084 17.68 6.17 0 15.20 0 1.11 0 0.47 0 0.00 0 30.44 0 433028 0 43 0 41.85 0 1.43 0 0.00 0 1.18 0 0.00 0 191.09 0 0.30 0 39280 0 636956 0 96831 0 7042 0 2996 0 0 0 193890 0 3 0 0 0 115 0 10478 0 196 0 10792 0 52.78 0 336197 0 3871 12075 3.119349005425 636956.0 433028.0 39280.0 96831.0 7042.0 2996.0 0.0 193890.0 336197.0 68.0 6.2 15.2 1.1 0.5 0.0 30.4 52.8 43 43 43.00 38 27389108 26.3 22.1 22.1 29.5 0.0 36.2 24.8 smartseq 1053339 SRR2088129 SRP060416 SRS980361 SRX1082098 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810564: T74_P3_F4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810564 GSM1810564 T74_P3_F4_ILC3 28812795 670065 2016-01-28 01:00:06 31864778 28812795 670065 1 670065 index:0,count:670065,average:43,stdev:0 GSM1810564_r1 2.4 5.34 0.22 18663098 22627687 14151263 18382471 121.24 129.9 0 0 0 0 0 0 56.3 75.29 863505 254974 863505 254974 63.18 71.63 863505 286123 863505 242585 2635770 14.12 6.23 0 17.04 0 1.07 0 0.39 0 0.00 0 30.96 0 452856 0 43 0 41.79 0 1.26 0 0.00 0 1.11 0 0.00 0 114.87 0 0.33 0 41741 0 670065 0 114200 0 7190 0 2589 0 0 0 207430 0 0 0 0 0 149 0 12592 0 177 0 12918 0 50.54 0 338656 0 4842 15201 3.139405204461 670065.0 452856.0 41741.0 114200.0 7190.0 2589.0 0.0 207430.0 338656.0 67.6 6.2 17.0 1.1 0.4 0.0 31.0 50.5 43 43 43.00 38 28812795 26.2 22.3 22.1 29.4 0.0 35.9 24.5 smartseq 1053452 SRR2088130 SRP060416 SRS980360 SRX1082099 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810565: T74_P3_G4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810565 GSM1810565 T74_P3_G4_NK 30814273 716611 2016-01-28 01:00:06 33905665 30814273 716611 1 716611 index:0,count:716611,average:43,stdev:0 GSM1810565_r1 3.04 4.99 0.24 22249881 27827283 17013521 22571858 125.07 132.67 0 0 0 0 0 0 58.26 76.99 997053 313087 997053 313087 65.91 73.23 997053 354251 997053 297800 2744086 12.33 4.85 0 18.25 0 1.07 0 0.43 0 0.00 0 23.50 0 537442 0 43 0 41.84 0 1.47 0 0.00 0 1.12 0 0.00 0 161.24 0 0.32 0 34731 0 716611 0 130791 0 7636 0 3109 0 0 0 168424 0 2 0 0 0 137 0 14930 0 217 0 15286 0 56.75 0 406651 0 5655 17694 3.128912466844 716611.0 537442.0 34731.0 130791.0 7636.0 3109.0 0.0 168424.0 406651.0 75.0 4.8 18.3 1.1 0.4 0.0 23.5 56.7 43 43 43.00 38 30814273 26.3 22.4 22.3 29.1 0.0 36.1 24.7 smartseq 1053467 SRR2088131 SRP060416 SRS980359 SRX1082100 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810566: T74_P3_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810566 GSM1810566 T74_P3_G9_NK 108980232 2534424 2016-01-28 01:00:06 118029420 108980232 2534424 1 2534424 index:0,count:2534424,average:43,stdev:0 GSM1810566_r1 4.16 4.46 0.16 84297865 108839593 67179936 90837210 129.11 135.21 0 0 0 0 0 0 63.53 80.34 3454220 1285482 3454220 1285482 69.84 75.93 3454220 1413160 3454220 1214882 9053862 10.74 3.85 0 16.71 0 0.91 0 0.44 0 0.00 0 18.81 0 2023484 0 43 0 41.99 0 1.46 0 0.00 0 1.12 0 0.00 0 350.92 0 0.31 0 97585 0 2534424 0 423482 0 23016 0 11139 0 0 0 476785 0 26 0 0 0 605 0 67892 0 660 0 69183 0 63.13 0 1600002 0 10222 80081 7.834181177852 2534424.0 2023484.0 97585.0 423482.0 23016.0 11139.0 0.0 476785.0 1600002.0 79.8 3.9 16.7 0.9 0.4 0.0 18.8 63.1 43 43 43.00 38 108980232 26.2 22.7 22.8 28.3 0.0 36.2 25.1 smartseq 1053484 SRR2088132 SRP060416 SRS980356 SRX1082101 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810567: T74_P3_H1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810567 GSM1810567 T74_P3_H1_ILC2 110239272 2563704 2016-01-28 01:00:06 120541143 110239272 2563704 1 2563704 index:0,count:2563704,average:43,stdev:0 GSM1810567_r1 0.9 5.38 0.25 72389918 85203644 56077811 70896934 117.7 126.43 0 0 0 0 0 0 55.18 72.26 3388521 969918 3388521 969918 60.13 68.62 3388521 1056875 3388521 920993 11557311 15.97 6.20 0 16.20 0 0.93 0 0.42 0 0.00 0 30.09 0 1757642 0 43 0 41.78 0 1.23 0 0.00 0 1.12 0 0.00 0 279.68 0 0.32 0 158866 0 2563704 0 415446 0 23881 0 10702 0 0 0 771479 0 10 0 0 0 427 0 42953 0 634 0 44024 0 52.35 0 1342196 0 6019 50261 8.350390430304 2563704.0 1757642.0 158866.0 415446.0 23881.0 10702.0 0.0 771479.0 1342196.0 68.6 6.2 16.2 0.9 0.4 0.0 30.1 52.4 43 43 43.00 38 110239272 26.4 22.1 22.1 29.5 0.0 35.9 24.3 smartseq 1053500 SRR2088133 SRP060416 SRS980357 SRX1082102 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810568: T74_P4_A8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810568 GSM1810568 T74_P4_A8_ILC3 54208724 1260668 2016-01-28 01:00:06 59460950 54208724 1260668 1 1260668 index:0,count:1260668,average:43,stdev:0 GSM1810568_r1 3.2 5.71 0.26 35630621 43148981 28188124 35823260 121.1 127.09 0 0 0 0 0 0 58.51 75.04 1504555 505595 1504555 505595 64.05 71.48 1504555 553452 1504555 481651 5078004 14.25 6.08 0 15.09 0 0.98 0 0.41 0 0.00 0 30.07 0 864052 0 43 0 41.84 0 1.30 0 0.00 0 1.12 0 0.00 0 252.13 0 0.31 0 76622 0 1260668 0 190265 0 12339 0 5219 0 0 0 379058 0 2 0 0 0 199 0 23581 0 290 0 24072 0 53.45 0 673787 0 5413 27132 5.012377609459 1260668.0 864052.0 76622.0 190265.0 12339.0 5219.0 0.0 379058.0 673787.0 68.5 6.1 15.1 1.0 0.4 0.0 30.1 53.4 43 43 43.00 38 54208724 26.1 22.2 22.2 29.5 0.0 36.0 24.6 smartseq 1053516 SRR2088134 SRP060416 SRS980358 SRX1082103 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810569: T74_P4_A9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810569 GSM1810569 T74_P4_A9_ILC3 74145631 1724317 2016-01-28 01:00:06 81175636 74145631 1724317 1 1724317 index:0,count:1724317,average:43,stdev:0 GSM1810569_r1 3.51 4.88 0.21 55510967 69147685 45761145 59221730 124.57 129.41 0 0 0 0 0 0 59.01 72.25 2100985 785885 2100985 785885 63.63 68.56 2100985 847432 2100985 745659 7917688 14.26 4.44 0 14.15 0 0.98 0 0.63 0 0.00 0 21.16 0 1331716 0 43 0 42.07 0 1.36 0 0.00 0 1.16 0 0.00 0 269.89 0 0.29 0 76563 0 1724317 0 244039 0 16977 0 10828 0 0 0 364796 0 19 0 0 0 356 0 42285 0 507 0 43167 0 63.08 0 1087677 0 10205 49051 4.806565409113 1724317.0 1331716.0 76563.0 244039.0 16977.0 10828.0 0.0 364796.0 1087677.0 77.2 4.4 14.2 1.0 0.6 0.0 21.2 63.1 43 43 43.00 38 74145631 26.6 22.1 22.1 29.1 0.0 36.1 25.2 smartseq 1053532 SRR2088135 SRP060416 SRS980355 SRX1082104 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810570: T74_P4_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810570 GSM1810570 T74_P4_B4_NK 66997139 1558073 2016-01-28 01:00:06 73051621 66997139 1558073 1 1558073 index:0,count:1558073,average:43,stdev:0 GSM1810570_r1 2.96 6.49 0.21 41157744 49113360 31943160 40523535 119.33 126.86 0 0 0 0 0 0 52.49 68.88 1809911 527490 1809911 527490 57.57 66.16 1809911 578620 1809911 506623 7049892 17.13 6.88 0 15.36 0 0.99 0 0.51 0 0.00 0 34.00 0 1005008 0 43 0 41.71 0 1.40 0 0.00 0 1.13 0 0.00 0 207.74 0 0.31 0 107134 0 1558073 0 239248 0 15349 0 7992 0 0 0 529724 0 0 0 0 0 256 0 20097 0 445 0 20798 0 49.15 0 765760 0 3885 24035 6.186615186615 1558073.0 1005008.0 107134.0 239248.0 15349.0 7992.0 0.0 529724.0 765760.0 64.5 6.9 15.4 1.0 0.5 0.0 34.0 49.1 43 43 43.00 38 66997139 26.5 21.7 21.7 30.2 0.0 36.1 24.5 smartseq 1053548 SRR2088136 SRP060416 SRS980354 SRX1082105 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810571: T74_P4_C3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810571 GSM1810571 T74_P4_C3_ILC2 29239226 679982 2016-01-28 01:00:06 32969119 29239226 679982 1 679982 index:0,count:679982,average:43,stdev:0 GSM1810571_r1 2.0 6.52 0.22 18257127 21788968 13546105 17351016 119.34 128.09 0 0 0 0 0 0 51.37 70.46 870488 229398 870488 229398 58.29 66.88 870488 260319 870488 217751 2957551 16.20 6.78 0 17.80 0 1.00 0 0.41 0 0.00 0 32.91 0 446587 0 43 0 41.61 0 1.42 0 0.00 0 1.14 0 0.00 0 144.00 0 0.37 0 46075 0 679982 0 121019 0 6816 0 2798 0 0 0 223781 0 5 0 0 0 121 0 10748 0 161 0 11035 0 47.88 0 325568 0 3196 12851 4.020963704631 679982.0 446587.0 46075.0 121019.0 6816.0 2798.0 0.0 223781.0 325568.0 65.7 6.8 17.8 1.0 0.4 0.0 32.9 47.9 43 43 43.00 38 29239226 26.5 21.5 21.8 30.2 0.0 35.3 23.6 smartseq 1053565 SRR2088137 SRP060416 SRS980352 SRX1082106 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810572: T74_P4_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810572 GSM1810572 T74_P4_D2_NK 54704901 1272207 2016-01-28 01:00:06 61679677 54704901 1272207 1 1272207 index:0,count:1272207,average:43,stdev:0 GSM1810572_r1 2.53 7.62 0.22 34503016 41551572 26371777 33670369 120.43 127.68 0 0 0 0 0 0 55.46 74.01 1526552 469778 1526552 469778 60.97 70.65 1526552 516407 1526552 448431 5462424 15.83 6.59 0 16.68 0 0.95 0 0.38 0 0.00 0 32.09 0 847015 0 43 0 41.55 0 1.44 0 0.00 0 1.15 0 0.00 0 241.05 0 0.37 0 83865 0 1272207 0 212251 0 12098 0 4868 0 0 0 408226 0 0 0 0 0 186 0 21440 0 368 0 21994 0 49.89 0 634764 0 4847 25262 5.211883639365 1272207.0 847015.0 83865.0 212251.0 12098.0 4868.0 0.0 408226.0 634764.0 66.6 6.6 16.7 1.0 0.4 0.0 32.1 49.9 43 43 43.00 38 54704901 26.4 21.3 21.6 30.8 0.0 35.1 23.3 smartseq 1053581 SRR2088138 SRP060416 SRS980351 SRX1082107 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810573: T74_P4_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810573 GSM1810573 T74_P4_E6_NK 149745264 3482448 2016-01-28 01:00:06 161674582 149745264 3482448 1 3482448 index:0,count:3482448,average:43,stdev:0 GSM1810573_r1 4.05 5.53 0.19 109714411 138435923 84405265 112122451 126.18 132.84 0 0 0 0 0 0 58.91 77.37 4804540 1558855 4804540 1558855 66.2 73.25 4804540 1751987 4804540 1475915 13338628 12.16 4.62 0 18.14 0 1.01 0 0.42 0 0.00 0 22.57 0 2646338 0 43 0 41.89 0 1.40 0 0.00 0 1.16 0 0.00 0 338.83 0 0.31 0 160744 0 3482448 0 631542 0 35173 0 14780 0 0 0 786157 0 24 0 0 0 630 0 80248 0 921 0 81823 0 57.86 0 2014796 0 8572 96426 11.248950069995 3482448.0 2646338.0 160744.0 631542.0 35173.0 14780.0 0.0 786157.0 2014796.0 76.0 4.6 18.1 1.0 0.4 0.0 22.6 57.9 43 43 43.00 38 149745264 26.3 22.4 22.4 28.9 0.0 36.1 24.8 smartseq 1053596 SRR2088139 SRP060416 SRS980353 SRX1082108 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810574: T74_P4_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810574 GSM1810574 T74_P4_E7_NK 39124668 909876 2016-01-28 01:00:06 42899615 39124668 909876 1 909876 index:0,count:909876,average:43,stdev:0 GSM1810574_r1 2.92 5.91 0.29 26913801 32526332 21424254 27130840 120.85 126.64 0 0 0 0 0 0 55.33 70.64 1096992 363599 1096992 363599 60.14 67.2 1096992 395192 1096992 345847 4488598 16.68 5.49 0 15.66 0 1.00 0 0.52 0 0.00 0 26.26 0 657159 0 43 0 41.63 0 1.32 0 0.00 0 1.11 0 0.00 0 192.68 0 0.32 0 49978 0 909876 0 142474 0 9075 0 4719 0 0 0 238923 0 1 0 0 0 276 0 14732 0 245 0 15254 0 56.57 0 514685 0 3135 17345 5.532695374801 909876.0 657159.0 49978.0 142474.0 9075.0 4719.0 0.0 238923.0 514685.0 72.2 5.5 15.7 1.0 0.5 0.0 26.3 56.6 43 43 43.00 38 39124668 27.1 21.5 21.6 29.8 0.0 36.0 24.5 smartseq 1053708 SRR2088140 SRP060416 SRS980349 SRX1082109 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810575: T74_P4_F5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810575 GSM1810575 T74_P4_F5_ILC2 28534843 663601 2016-01-28 01:00:06 31387046 28534843 663601 1 663601 index:0,count:663601,average:43,stdev:0 GSM1810575_r1 2.57 5.51 0.23 19713513 24601371 15692869 20588969 124.79 131.2 0 0 0 0 0 0 58.89 74.82 823476 279867 823476 279867 64.6 71.26 823476 306975 823476 266540 2550172 12.94 5.57 0 15.24 0 0.89 0 0.42 0 0.00 0 27.07 0 475201 0 43 0 41.96 0 1.39 0 0.00 0 1.14 0 0.00 0 199.08 0 0.30 0 36963 0 663601 0 101163 0 5914 0 2819 0 0 0 179667 0 11 0 0 0 103 0 12868 0 151 0 13133 0 56.36 0 374038 0 3922 14830 3.781234064253 663601.0 475201.0 36963.0 101163.0 5914.0 2819.0 0.0 179667.0 374038.0 71.6 5.6 15.2 0.9 0.4 0.0 27.1 56.4 43 43 43.00 38 28534843 26.4 22.2 22.2 29.2 0.0 36.1 25.0 smartseq 1053724 SRR2088141 SRP060416 SRS980350 SRX1082110 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810576: T74_P4_G4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810576 GSM1810576 T74_P4_G4_NK 224432996 5219372 2016-01-28 01:00:06 241701693 224432996 5219372 1 5219372 index:0,count:5219372,average:43,stdev:0 GSM1810576_r1 2.05 4.05 0.2 156408643 187181733 118878508 153624842 119.67 129.23 0 0 0 0 0 0 55.68 74.08 7637022 2103237 7637022 2103237 61.35 70.0 7637022 2317507 7637022 1987298 20914438 13.37 5.26 0 17.98 0 1.13 0 0.52 0 0.00 0 25.97 0 3777572 0 43 0 41.87 0 1.33 0 0.00 0 1.20 0 0.00 0 323.96 0 0.31 0 274334 0 5219372 0 938395 0 58857 0 27343 0 0 0 1355600 0 20 0 0 0 951 0 98110 0 1485 0 100566 0 54.40 0 2839177 0 7531 116869 15.518390651972 5219372.0 3777572.0 274334.0 938395.0 58857.0 27343.0 0.0 1355600.0 2839177.0 72.4 5.3 18.0 1.1 0.5 0.0 26.0 54.4 43 43 43.00 38 224432996 26.1 22.5 22.6 28.8 0.0 36.0 24.7 smartseq 1053741 SRR2088142 SRP060416 SRS980348 SRX1082111 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810577: T74_P4_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810577 GSM1810577 T74_P4_G9_NK 125605967 2921069 2016-01-28 01:00:06 135816500 125605967 2921069 1 2921069 index:0,count:2921069,average:43,stdev:0 GSM1810577_r1 2.76 5.72 0.24 84228820 102905628 64276740 84016489 122.17 130.71 0 0 0 0 0 0 53.75 71.41 3897845 1097226 3897845 1097226 59.98 67.93 3897845 1224596 3897845 1043745 12640876 15.01 5.80 0 17.29 0 1.01 0 0.45 0 0.00 0 28.65 0 2041510 0 43 0 41.83 0 1.35 0 0.00 0 1.14 0 0.00 0 269.64 0 0.30 0 169296 0 2921069 0 504993 0 29467 0 13180 0 0 0 836912 0 17 0 0 0 500 0 51476 0 806 0 52799 0 52.60 0 1536517 0 6327 61418 9.707286233602 2921069.0 2041510.0 169296.0 504993.0 29467.0 13180.0 0.0 836912.0 1536517.0 69.9 5.8 17.3 1.0 0.5 0.0 28.7 52.6 43 43 43.00 38 125605967 26.0 22.2 22.1 29.6 0.0 36.1 24.6 smartseq 1053756 SRR2088143 SRP060416 SRS980347 SRX1082112 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810578: T74_P4_H1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810578 GSM1810578 T74_P4_H1_ILC3 133885832 3113624 2016-01-28 01:00:06 147171046 133885832 3113624 1 3113624 index:0,count:3113624,average:43,stdev:0 GSM1810578_r1 1.84 6.09 0.25 85469002 99808007 66636666 83011219 116.78 124.57 0 0 0 0 0 0 52.52 68.5 3858007 1094896 3858007 1094896 56.64 64.77 3858007 1180673 3858007 1035245 15336321 17.94 6.40 0 15.62 0 1.16 0 0.54 0 0.00 0 31.35 0 2084545 0 43 0 41.69 0 1.41 0 0.00 0 1.17 0 0.00 0 243.67 0 0.33 0 199298 0 3113624 0 486257 0 35977 0 16951 0 0 0 976151 0 41 0 0 0 671 0 51352 0 1069 0 53133 0 51.33 0 1598288 0 5721 59495 10.399405698304 3113624.0 2084545.0 199298.0 486257.0 35977.0 16951.0 0.0 976151.0 1598288.0 66.9 6.4 15.6 1.2 0.5 0.0 31.4 51.3 43 43 43.00 38 133885832 26.2 21.9 21.8 30.1 0.0 35.7 24.1 smartseq 1053771 SRR2088144 SRP060416 SRS980345 SRX1082113 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810579: T74_P4_H3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810579 GSM1810579 T74_P4_H3_ILC3 141626262 3293634 2016-01-28 01:00:06 154525194 141626262 3293634 1 3293634 index:0,count:3293634,average:43,stdev:0 GSM1810579_r1 3.04 6.01 0.16 91498642 109704369 70894828 89680593 119.9 126.5 0 0 0 0 0 0 55.85 73.14 4036191 1240882 4036191 1240882 61.42 69.34 4036191 1364573 4036191 1176365 13367496 14.61 6.43 0 15.95 0 0.96 0 0.47 0 0.00 0 31.12 0 2221812 0 43 0 41.79 0 1.39 0 0.00 0 1.17 0 0.00 0 50.46 0 0.33 0 211656 0 3293634 0 525248 0 31540 0 15343 0 0 0 1024939 0 14 0 0 0 585 0 58494 0 788 0 59881 0 51.51 0 1696564 0 6331 69418 10.964776496604 3293634.0 2221812.0 211656.0 525248.0 31540.0 15343.0 0.0 1024939.0 1696564.0 67.5 6.4 15.9 1.0 0.5 0.0 31.1 51.5 43 43 43.00 38 141626262 26.2 22.2 22.2 29.5 0.0 35.8 24.3 smartseq 1053787 SRR2088145 SRP060416 SRS980344 SRX1082114 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810580: T74_P4_H4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810580 GSM1810580 T74_P4_H4_ILC3 113666243 2643401 2016-01-28 01:00:06 124526841 113666243 2643401 1 2643401 index:0,count:2643401,average:43,stdev:0 GSM1810580_r1 2.17 6.08 0.19 70683624 81792971 55084431 67594282 115.72 122.71 0 0 0 0 0 0 52.29 68.28 3157153 901331 3157153 901331 57.37 65.43 3157153 988828 3157153 863774 12974111 18.36 6.81 0 15.26 0 0.94 0 0.40 0 0.00 0 33.45 0 1723584 0 43 0 41.73 0 1.39 0 0.00 0 1.17 0 0.00 0 257.20 0 0.32 0 179962 0 2643401 0 403469 0 24969 0 10575 0 0 0 884273 0 2 0 0 0 487 0 38227 0 753 0 39469 0 49.94 0 1320115 0 4671 44439 9.513808606294 2643401.0 1723584.0 179962.0 403469.0 24969.0 10575.0 0.0 884273.0 1320115.0 65.2 6.8 15.3 0.9 0.4 0.0 33.5 49.9 43 43 43.00 38 113666243 26.5 21.7 21.6 30.2 0.0 35.8 24.2 smartseq 1053800 SRR2088146 SRP060416 SRS980343 SRX1082115 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810581: T74_P4_H5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810581 GSM1810581 T74_P4_H5_ILC3 65720641 1528387 2016-01-28 01:00:06 71744689 65720641 1528387 1 1528387 index:0,count:1528387,average:43,stdev:0 GSM1810581_r1 3.04 5.97 0.26 45744504 56966904 37832541 48958483 124.53 129.41 0 0 0 0 0 0 59.14 72.4 1722328 653744 1722328 653744 62.77 68.41 1722328 693926 1722328 617771 6847725 14.97 5.34 0 13.24 0 0.96 0 0.53 0 0.00 0 26.18 0 1105419 0 43 0 41.90 0 1.43 0 0.00 0 1.15 0 0.00 0 262.01 0 0.30 0 81683 0 1528387 0 202421 0 14641 0 8173 0 0 0 400154 0 3 0 0 0 254 0 32595 0 461 0 33313 0 59.08 0 902998 0 6622 36760 5.551192993053 1528387.0 1105419.0 81683.0 202421.0 14641.0 8173.0 0.0 400154.0 902998.0 72.3 5.3 13.2 1.0 0.5 0.0 26.2 59.1 43 43 43.00 38 65720641 26.5 22.1 22.0 29.4 0.0 36.2 25.0 smartseq 1053817 SRR2088147 SRP060416 SRS980342 SRX1082116 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810582: T74_P4_H6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810582 GSM1810582 T74_P4_H6_ILC3 49158374 1143218 2016-01-28 01:00:06 53845638 49158374 1143218 1 1143218 index:0,count:1143218,average:43,stdev:0 GSM1810582_r1 4.08 4.73 0.17 38836898 47983322 31019841 40204104 123.55 129.61 0 0 0 0 0 0 58.92 74.42 1601159 548169 1601159 548169 64.74 70.61 1601159 602328 1601159 520062 5159251 13.28 3.61 0 16.96 0 0.95 0 0.55 0 0.00 0 17.12 0 930426 0 43 0 42.12 0 1.34 0 0.00 0 1.15 0 0.00 0 274.37 0 0.29 0 41300 0 1143218 0 193885 0 10830 0 6287 0 0 0 195675 0 2 0 0 0 288 0 26128 0 323 0 26741 0 64.43 0 736541 0 7078 30781 4.348827352359 1143218.0 930426.0 41300.0 193885.0 10830.0 6287.0 0.0 195675.0 736541.0 81.4 3.6 17.0 0.9 0.5 0.0 17.1 64.4 43 43 43.00 38 49158374 26.9 21.9 21.8 29.4 0.0 36.2 25.2 smartseq 1053848 SRR2088149 SRP060416 SRS980340 SRX1082118 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810584: T74_P4_H9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810584 GSM1810584 T74_P4_H9_ILC3 131806782 3065274 2016-01-28 01:00:06 141748866 131806782 3065274 1 3065274 index:0,count:3065274,average:43,stdev:0 GSM1810584_r1 3.16 6.01 0.23 88288885 108976691 69189766 89804253 123.43 129.79 0 0 0 0 0 0 59.54 77.02 3702786 1274029 3702786 1274029 65.57 73.17 3702786 1403099 3702786 1210296 11391673 12.90 5.91 0 15.85 0 0.89 0 0.43 0 0.00 0 28.87 0 2139907 0 43 0 41.83 0 1.39 0 0.00 0 1.12 0 0.00 0 306.53 0 0.31 0 181121 0 3065274 0 485810 0 27179 0 13278 0 0 0 884910 0 14 0 0 0 772 0 61829 0 850 0 63465 0 53.96 0 1654097 0 7357 72752 9.888813375017 3065274.0 2139907.0 181121.0 485810.0 27179.0 13278.0 0.0 884910.0 1654097.0 69.8 5.9 15.8 0.9 0.4 0.0 28.9 54.0 43 43 43.00 38 131806782 26.0 22.3 22.2 29.5 0.0 36.2 24.7 smartseq 1053960 SRR2088150 SRP060416 SRS980346 SRX1082119 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810585: T75_P1_A10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810585 GSM1810585 T75_P1_A10_ILC3 21150711 491877 2016-01-28 01:00:06 24546707 21150711 491877 1 491877 index:0,count:491877,average:43,stdev:0 GSM1810585_r1 2.91 8.07 0.18 14692545 17638639 11624083 14692015 120.05 126.39 0 0 0 0 0 0 55.68 71.83 602467 201079 602467 201079 59.96 68.55 602467 216538 602467 191912 2577277 17.54 5.18 0 16.51 0 1.16 0 0.47 0 0.00 0 24.94 0 361146 0 43 0 41.52 0 1.39 0 0.00 0 1.12 0 0.00 0 104.16 0 0.38 0 25469 0 491877 0 81197 0 5706 0 2329 0 0 0 122696 0 0 0 0 0 80 0 8725 0 216 0 9021 0 56.91 0 279949 0 2650 10119 3.818490566038 491877.0 361146.0 25469.0 81197.0 5706.0 2329.0 0.0 122696.0 279949.0 73.4 5.2 16.5 1.2 0.5 0.0 24.9 56.9 43 43 43.00 38 21150711 27.5 20.1 20.9 31.5 0.0 34.6 22.9 smartseq 1053977 SRR2088151 SRP060416 SRS980338 SRX1082120 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810586: T75_P1_A11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810586 GSM1810586 T75_P1_A11_ILC3 50421542 1172594 2016-01-28 01:00:06 57087868 50421542 1172594 1 1172594 index:0,count:1172594,average:43,stdev:0 GSM1810586_r1 2.22 9.74 0.24 29376593 34189182 22501322 27577619 116.38 122.56 0 0 0 0 0 0 51.98 69.75 1258016 381164 1258016 381164 56.33 66.94 1258016 413123 1258016 365791 5657453 19.26 7.24 0 15.94 0 1.19 0 0.33 0 0.00 0 35.94 0 733333 0 43 0 41.18 0 1.43 0 0.00 0 1.19 0 0.00 0 50.86 0 0.35 0 84946 0 1172594 0 186888 0 13927 0 3865 0 0 0 421469 0 27 0 0 0 129 0 14478 0 534 0 15168 0 46.60 0 546445 0 2351 16067 6.834113143343 1172594.0 733333.0 84946.0 186888.0 13927.0 3865.0 0.0 421469.0 546445.0 62.5 7.2 15.9 1.2 0.3 0.0 35.9 46.6 43 43 43.00 38 50421542 26.7 20.6 20.5 32.2 0.0 35.2 23.2 smartseq 1053993 SRR2088152 SRP060416 SRS980337 SRX1082121 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810587: T75_P1_A12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810587 GSM1810587 T75_P1_A12_ILC3 23766444 552708 2016-01-28 01:00:06 27690604 23766444 552708 1 552708 index:0,count:552708,average:43,stdev:0 GSM1810587_r1 4.2 9.4 0.29 15777562 18894073 11711441 14827011 119.75 126.6 0 0 0 0 0 0 51.55 71.24 698648 202966 698648 202966 58.49 69.43 698648 230279 698648 197804 2771797 17.57 5.69 0 19.69 0 1.38 0 0.37 0 0.00 0 27.01 0 393713 0 43 0 41.11 0 1.36 0 0.00 0 1.13 0 0.00 0 110.54 0 0.42 0 31467 0 552708 0 108809 0 7632 0 2052 0 0 0 149311 0 7 0 0 0 88 0 8828 0 254 0 9177 0 51.55 0 284904 0 1771 9674 5.462450592885 552708.0 393713.0 31467.0 108809.0 7632.0 2052.0 0.0 149311.0 284904.0 71.2 5.7 19.7 1.4 0.4 0.0 27.0 51.5 43 43 43.00 38 23766444 27.2 19.6 20.3 32.9 0.0 34.3 22.2 smartseq 1054009 SRR2088153 SRP060416 SRS979768 SRX1082122 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810588: T75_P1_A1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810588 GSM1810588 T75_P1_A1_ILC3 50158425 1166475 2016-01-28 01:00:06 57742178 50158425 1166475 1 1166475 index:0,count:1166475,average:43,stdev:0 GSM1810588_r1 2.64 8.2 0.23 33197642 39111261 25307732 31442579 117.81 124.24 0 0 0 0 0 0 53.25 71.36 1474518 435260 1474518 435260 58.81 68.24 1474518 480650 1474518 416231 5808064 17.50 5.86 0 17.78 0 1.10 0 0.40 0 0.00 0 28.43 0 817361 0 43 0 41.49 0 1.38 0 0.00 0 1.19 0 0.00 0 247.02 0 0.37 0 68329 0 1166475 0 207378 0 12798 0 4629 0 0 0 331687 0 6 0 0 0 177 0 18648 0 556 0 19387 0 52.29 0 609983 0 4399 21408 4.866560581950 1166475.0 817361.0 68329.0 207378.0 12798.0 4629.0 0.0 331687.0 609983.0 70.1 5.9 17.8 1.1 0.4 0.0 28.4 52.3 43 43 43.00 38 50158425 26.6 20.6 21.1 31.7 0.0 34.7 22.9 smartseq 1054025 SRR2088154 SRP060416 SRS980339 SRX1082123 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810589: T75_P1_A3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810589 GSM1810589 T75_P1_A3_ILC3 107165675 2492225 2016-01-28 01:00:06 118728006 107165675 2492225 1 2492225 index:0,count:2492225,average:43,stdev:0 GSM1810589_r1 3.57 7.04 0.38 68624965 81999324 53030403 66754676 119.49 125.88 0 0 0 0 0 0 51.76 68.26 2973677 868470 2973677 868470 57.17 65.4 2973677 959361 2973677 832125 12649871 18.43 6.21 0 16.27 0 1.11 0 0.48 0 0.00 0 31.09 0 1677949 0 43 0 41.68 0 1.27 0 0.00 0 1.18 0 0.00 0 271.88 0 0.32 0 154729 0 2492225 0 405596 0 27663 0 11884 0 0 0 774729 0 11 0 0 0 442 0 34167 0 1025 0 35645 0 51.05 0 1272353 0 4022 39032 9.704624564893 2492225.0 1677949.0 154729.0 405596.0 27663.0 11884.0 0.0 774729.0 1272353.0 67.3 6.2 16.3 1.1 0.5 0.0 31.1 51.1 43 43 43.00 38 107165675 26.3 21.5 21.5 30.8 0.0 35.7 24.2 smartseq 1054041 SRR2088155 SRP060416 SRS980336 SRX1082124 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810590: T75_P1_A5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810590 GSM1810590 T75_P1_A5_ILC3 76548471 1780197 2016-01-28 01:00:06 86128056 76548471 1780197 1 1780197 index:0,count:1780197,average:43,stdev:0 GSM1810590_r1 3.88 6.5 0.22 52901637 64971960 42573781 54488647 122.82 127.99 0 0 0 0 0 0 58.0 73.24 2095845 746710 2095845 746710 62.31 69.56 2095845 802233 2095845 709159 8365859 15.81 5.36 0 15.05 0 0.96 0 0.40 0 0.00 0 26.33 0 1287500 0 43 0 41.76 0 1.35 0 0.00 0 1.15 0 0.00 0 267.03 0 0.32 0 95482 0 1780197 0 267943 0 17013 0 7032 0 0 0 468652 0 8 0 0 0 328 0 34779 0 664 0 35779 0 57.27 0 1019557 0 5910 39669 6.712182741117 1780197.0 1287500.0 95482.0 267943.0 17013.0 7032.0 0.0 468652.0 1019557.0 72.3 5.4 15.1 1.0 0.4 0.0 26.3 57.3 43 43 43.00 38 76548471 26.5 21.5 21.6 30.3 0.0 35.4 24.1 smartseq 1054057 SRR2088156 SRP060416 SRS980335 SRX1082125 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810591: T75_P1_A7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810591 GSM1810591 T75_P1_A7_ILC3 24180921 562347 2016-01-28 01:00:06 28267962 24180921 562347 1 562347 index:0,count:562347,average:43,stdev:0 GSM1810591_r1 4.01 8.67 0.31 17565783 21323350 13841626 17577850 121.39 126.99 0 0 0 0 0 0 55.46 71.83 710903 239930 710903 239930 60.24 68.83 710903 260621 710903 229904 3279025 18.67 4.46 0 17.53 0 1.28 0 0.51 0 0.00 0 21.28 0 432615 0 43 0 41.44 0 1.33 0 0.00 0 1.14 0 0.01 0 101.22 0 0.42 0 25080 0 562347 0 98579 0 7181 0 2894 0 0 0 119657 0 8 0 0 0 78 0 10362 0 266 0 10714 0 59.40 0 334036 0 2695 11499 4.266790352505 562347.0 432615.0 25080.0 98579.0 7181.0 2894.0 0.0 119657.0 334036.0 76.9 4.5 17.5 1.3 0.5 0.0 21.3 59.4 43 43 43.00 38 24180921 27.4 19.8 21.0 31.9 0.0 34.3 22.4 smartseq 1054073 SRR2088157 SRP060416 SRS980334 SRX1082126 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810592: T75_P1_A8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810592 GSM1810592 T75_P1_A8_ILC3 38619590 898130 2016-01-28 01:00:06 43693301 38619590 898130 1 898130 index:0,count:898130,average:43,stdev:0 GSM1810592_r1 2.81 6.9 0.33 27917401 34627762 22419551 28876371 124.04 128.8 0 0 0 0 0 0 54.98 69.58 1089579 374260 1089579 374260 60.15 66.38 1089579 409499 1089579 357030 5042490 18.06 4.58 0 15.91 0 1.17 0 0.50 0 0.00 0 22.53 0 680768 0 43 0 41.68 0 1.38 0 0.00 0 1.18 0 0.01 0 248.71 0 0.34 0 41172 0 898130 0 142895 0 10500 0 4527 0 0 0 202335 0 0 0 0 0 156 0 15457 0 373 0 15986 0 59.89 0 537873 0 2329 17931 7.699012451696 898130.0 680768.0 41172.0 142895.0 10500.0 4527.0 0.0 202335.0 537873.0 75.8 4.6 15.9 1.2 0.5 0.0 22.5 59.9 43 43 43.00 38 38619590 27.8 20.6 20.7 30.9 0.0 35.4 24.0 smartseq 1054090 SRR2088158 SRP060416 SRS980333 SRX1082127 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810593: T75_P1_B12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810593 GSM1810593 T75_P1_B12_ILC3 80685974 1876418 2016-01-28 01:00:06 89856108 80685974 1876418 1 1876418 index:0,count:1876418,average:43,stdev:0 GSM1810593_r1 3.33 7.65 0.35 52837038 64981950 41465078 53373818 122.99 128.72 0 0 0 0 0 0 55.21 71.74 2107593 719241 2107593 719241 60.5 68.81 2107593 788090 2107593 689838 8493066 16.07 5.83 0 15.99 0 1.23 0 0.50 0 0.00 0 28.84 0 1302679 0 43 0 41.36 0 1.31 0 0.00 0 1.22 0 0.01 0 259.81 0 0.36 0 109356 0 1876418 0 300116 0 23160 0 9475 0 0 0 541104 0 26 0 0 0 383 0 29846 0 944 0 31199 0 53.43 0 1002563 0 2627 34558 13.154929577465 1876418.0 1302679.0 109356.0 300116.0 23160.0 9475.0 0.0 541104.0 1002563.0 69.4 5.8 16.0 1.2 0.5 0.0 28.8 53.4 43 43 43.00 38 80685974 27.1 21.1 20.9 30.9 0.0 35.6 23.8 smartseq 1054106 SRR2088159 SRP060416 SRS980332 SRX1082128 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810594: T75_P1_B1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810594 GSM1810594 T75_P1_B1_ILC3 164845015 3833605 2016-01-28 01:00:06 180088200 164845015 3833605 1 3833605 index:0,count:3833605,average:43,stdev:0 GSM1810594_r1 3.65 6.11 0.23 112360521 139105394 88309868 114979402 123.8 130.2 0 0 0 0 0 0 57.78 74.6 4715787 1577531 4715787 1577531 63.13 70.8 4715787 1723741 4715787 1497039 16676285 14.84 5.48 0 16.06 0 0.96 0 0.46 0 0.00 0 27.36 0 2730395 0 43 0 41.76 0 1.36 0 0.00 0 1.12 0 0.00 0 293.64 0 0.30 0 210152 0 3833605 0 615804 0 36936 0 17473 0 0 0 1048801 0 11 0 0 0 686 0 77150 0 1472 0 79319 0 55.16 0 2114591 0 6863 90034 13.118752732041 3833605.0 2730395.0 210152.0 615804.0 36936.0 17473.0 0.0 1048801.0 2114591.0 71.2 5.5 16.1 1.0 0.5 0.0 27.4 55.2 43 43 43.00 38 164845015 26.3 21.9 21.8 30.0 0.0 35.9 24.7 smartseq 1054219 SRR2088160 SRP060416 SRS980331 SRX1082129 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810595: T75_P1_B3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810595 GSM1810595 T75_P1_B3_ILC3 144665330 3364310 2016-01-28 01:00:06 158785293 144665330 3364310 1 3364310 index:0,count:3364310,average:43,stdev:0 GSM1810595_r1 5.58 6.63 0.25 99564858 127050030 77342349 103292403 127.61 133.55 0 0 0 0 0 0 59.68 77.93 4102937 1442821 4102937 1442821 66.09 73.57 4102937 1597633 4102937 1362182 13112661 13.17 5.36 0 16.82 0 0.94 0 0.43 0 0.00 0 26.77 0 2417535 0 43 0 41.77 0 1.31 0 0.00 0 1.17 0 0.00 0 318.72 0 0.31 0 180418 0 3364310 0 566035 0 31689 0 14351 0 0 0 900735 0 37 0 0 0 675 0 72767 0 1317 0 74796 0 55.03 0 1851500 0 7168 85308 11.901227678571 3364310.0 2417535.0 180418.0 566035.0 31689.0 14351.0 0.0 900735.0 1851500.0 71.9 5.4 16.8 0.9 0.4 0.0 26.8 55.0 43 43 43.00 38 144665330 26.2 22.0 21.9 29.9 0.0 35.8 24.7 smartseq 1054234 SRR2088161 SRP060416 SRS980330 SRX1082130 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810596: T75_P1_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810596 GSM1810596 T75_P1_B4_NK 113535007 2640349 2016-01-28 01:00:06 125025324 113535007 2640349 1 2640349 index:0,count:2640349,average:43,stdev:0 GSM1810596_r1 2.66 6.84 0.21 74159638 92611811 56253809 74661598 124.88 132.72 0 0 0 0 0 0 55.61 74.52 3321780 1006302 3321780 1006302 62.54 70.74 3321780 1131649 3321780 955188 10958803 14.78 5.96 0 17.39 0 1.20 0 0.41 0 0.00 0 29.86 0 1809448 0 43 0 41.66 0 1.43 0 0.00 0 1.19 0 0.00 0 279.57 0 0.31 0 157432 0 2640349 0 459132 0 31691 0 10701 0 0 0 788509 0 19 0 0 0 469 0 49155 0 1000 0 50643 0 51.14 0 1350316 0 4916 59281 12.058787632221 2640349.0 1809448.0 157432.0 459132.0 31691.0 10701.0 0.0 788509.0 1350316.0 68.5 6.0 17.4 1.2 0.4 0.0 29.9 51.1 43 43 43.00 38 113535007 26.2 21.9 21.9 30.0 0.0 35.8 24.5 smartseq 1054251 SRR2088162 SRP060416 SRS980329 SRX1082131 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810597: T75_P1_B5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810597 GSM1810597 T75_P1_B5_ILC3 134469385 3127195 2016-01-28 01:00:06 147698078 134469385 3127195 1 3127195 index:0,count:3127195,average:43,stdev:0 GSM1810597_r1 4.87 6.82 0.27 87095740 106947940 68049179 87352525 122.79 128.37 0 0 0 0 0 0 60.56 78.89 3589163 1289649 3589163 1289649 66.48 75.33 3589163 1415547 3589163 1231351 11640547 13.37 6.08 0 15.82 0 1.04 0 0.30 0 0.00 0 30.57 0 2129428 0 43 0 41.63 0 1.44 0 0.00 0 1.16 0 0.00 0 288.66 0 0.31 0 190233 0 3127195 0 494770 0 32400 0 9305 0 0 0 956062 0 3 0 0 0 545 0 57325 0 1241 0 59114 0 52.27 0 1634658 0 5372 67240 12.516753536858 3127195.0 2129428.0 190233.0 494770.0 32400.0 9305.0 0.0 956062.0 1634658.0 68.1 6.1 15.8 1.0 0.3 0.0 30.6 52.3 43 43 43.00 38 134469385 26.3 21.7 21.6 30.4 0.0 35.8 24.4 smartseq 1054267 SRR2088163 SRP060416 SRS980328 SRX1082132 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810598: T75_P1_B6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810598 GSM1810598 T75_P1_B6_ILC3 120437926 2800882 2016-01-28 01:00:06 134164732 120437926 2800882 1 2800882 index:0,count:2800882,average:43,stdev:0 GSM1810598_r1 6.17 6.09 0.38 85351211 107430355 68396668 89438265 125.87 130.76 0 0 0 0 0 0 56.15 71.11 3353777 1163385 3353777 1163385 60.53 67.58 3353777 1254172 3353777 1105699 13592398 15.93 4.94 0 15.56 0 1.12 0 0.60 0 0.00 0 24.30 0 2072004 0 43 0 41.81 0 1.29 0 0.00 0 1.13 0 0.00 0 288.09 0 0.33 0 138471 0 2800882 0 435917 0 31462 0 16839 0 0 0 680577 0 4 0 0 0 429 0 45999 0 1088 0 47520 0 58.41 0 1636087 0 5469 52507 9.600841104407 2800882.0 2072004.0 138471.0 435917.0 31462.0 16839.0 0.0 680577.0 1636087.0 74.0 4.9 15.6 1.1 0.6 0.0 24.3 58.4 43 43 43.00 38 120437926 26.8 21.4 21.5 30.3 0.0 35.5 24.3 smartseq 1054283 SRR2088164 SRP060416 SRS980326 SRX1082133 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810599: T75_P1_B7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810599 GSM1810599 T75_P1_B7_ILC3 108477734 2522738 2016-01-28 01:00:06 119704473 108477734 2522738 1 2522738 index:0,count:2522738,average:43,stdev:0 GSM1810599_r1 1.43 9.35 0.23 59411096 68357632 44851024 55090526 115.06 122.83 0 0 0 0 0 0 51.1 69.64 2688287 759973 2688287 759973 56.05 67.85 2688287 833595 2688287 740484 12489810 21.02 7.83 0 15.69 0 1.33 0 0.39 0 0.00 0 39.34 0 1487107 0 43 0 41.10 0 1.41 0 0.01 0 1.16 0 0.00 0 252.27 0 0.34 0 197585 0 2522738 0 395820 0 33431 0 9717 0 0 0 992483 0 1 0 0 0 430 0 33316 0 1191 0 34938 0 43.26 0 1091287 0 2015 37360 18.540942928040 2522738.0 1487107.0 197585.0 395820.0 33431.0 9717.0 0.0 992483.0 1091287.0 58.9 7.8 15.7 1.3 0.4 0.0 39.3 43.3 43 43 43.00 38 108477734 26.7 20.9 20.7 31.7 0.0 35.6 23.6 smartseq 1054298 SRR2088165 SRP060416 SRS980327 SRX1082134 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810600: T75_P1_B8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810600 GSM1810600 T75_P1_B8_ILC3 32418517 753919 2016-01-28 01:00:06 37398577 32418517 753919 1 753919 index:0,count:753919,average:43,stdev:0 GSM1810600_r1 5.28 8.95 0.4 21033708 25354613 16005346 20081362 120.54 125.47 0 0 0 0 0 0 53.4 71.81 879206 278123 879206 278123 59.56 68.56 879206 310169 879206 265506 3409850 16.21 5.89 0 17.71 0 1.31 0 0.44 0 0.00 0 29.17 0 520808 0 43 0 41.33 0 1.37 0 0.01 0 1.19 0 0.00 0 159.65 0 0.40 0 44438 0 753919 0 133519 0 9888 0 3324 0 0 0 219899 0 16 0 0 0 89 0 10263 0 347 0 10715 0 51.37 0 387289 0 2092 12789 6.113288718929 753919.0 520808.0 44438.0 133519.0 9888.0 3324.0 0.0 219899.0 387289.0 69.1 5.9 17.7 1.3 0.4 0.0 29.2 51.4 43 43 43.00 38 32418517 27.4 20.2 20.8 31.6 0.0 34.7 22.9 smartseq 1054316 SRR2088166 SRP060416 SRS980325 SRX1082135 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810601: T75_P1_C10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810601 GSM1810601 T75_P1_C10_ILC3 44018541 1023687 2016-01-28 01:00:06 49832300 44018541 1023687 1 1023687 index:0,count:1023687,average:43,stdev:0 GSM1810601_r1 2.96 7.68 0.27 28138805 34603058 21940146 28399766 122.97 129.44 0 0 0 0 0 0 54.87 71.8 1174016 379758 1174016 379758 60.34 68.75 1174016 417635 1174016 363587 4803917 17.07 6.24 0 15.94 0 1.16 0 0.42 0 0.00 0 30.82 0 692080 0 43 0 41.48 0 1.47 0 0.00 0 1.15 0 0.00 0 193.96 0 0.35 0 63895 0 1023687 0 163201 0 11827 0 4288 0 0 0 315492 0 4 0 0 0 132 0 17932 0 428 0 18496 0 51.66 0 528879 0 3199 20848 6.517036573929 1023687.0 692080.0 63895.0 163201.0 11827.0 4288.0 0.0 315492.0 528879.0 67.6 6.2 15.9 1.2 0.4 0.0 30.8 51.7 43 43 43.00 38 44018541 27.0 21.0 21.1 31.0 0.0 35.3 23.8 smartseq 1054329 SRR2088167 SRP060416 SRS980324 SRX1082136 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810602: T75_P1_C11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810602 GSM1810602 T75_P1_C11_ILC3 70913880 1649160 2016-01-28 01:00:06 78555335 70913880 1649160 1 1649160 index:0,count:1649160,average:43,stdev:0 GSM1810602_r1 2.65 8.3 0.28 41779078 48614840 31531518 38752068 116.36 122.9 0 0 0 0 0 0 52.16 70.84 1859726 542219 1859726 542219 57.48 67.85 1859726 597544 1859726 519375 7901264 18.91 7.06 0 16.62 0 1.38 0 0.36 0 0.00 0 35.23 0 1039513 0 43 0 41.19 0 1.32 0 0.00 0 1.15 0 0.00 0 212.03 0 0.33 0 116364 0 1649160 0 274085 0 22736 0 5936 0 0 0 580975 0 3 0 0 0 201 0 24100 0 852 0 25156 0 46.41 0 765428 0 1943 26408 13.591353576943 1649160.0 1039513.0 116364.0 274085.0 22736.0 5936.0 0.0 580975.0 765428.0 63.0 7.1 16.6 1.4 0.4 0.0 35.2 46.4 43 43 43.00 38 70913880 26.9 20.8 20.5 31.8 0.0 35.7 23.8 smartseq 1054345 SRR2088168 SRP060416 SRS980323 SRX1082137 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810603: T75_P1_C1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810603 GSM1810603 T75_P1_C1_ILC3 146596288 3409216 2016-01-28 01:00:06 160262566 146596288 3409216 1 3409216 index:0,count:3409216,average:43,stdev:0 GSM1810603_r1 3.49 6.33 0.19 98364937 122951057 77216272 102136130 124.99 132.27 0 0 0 0 0 0 57.66 74.65 4181137 1381690 4181137 1381690 63.14 71.4 4181137 1512847 4181137 1321605 15185793 15.44 5.64 0 15.99 0 1.04 0 0.44 0 0.00 0 28.23 0 2396131 0 43 0 41.72 0 1.39 0 0.00 0 1.12 0 0.00 0 395.91 0 0.30 0 192292 0 3409216 0 545122 0 35479 0 15104 0 0 0 962502 0 54 0 0 0 553 0 58864 0 1243 0 60714 0 54.29 0 1851009 0 5236 68265 13.037624140565 3409216.0 2396131.0 192292.0 545122.0 35479.0 15104.0 0.0 962502.0 1851009.0 70.3 5.6 16.0 1.0 0.4 0.0 28.2 54.3 43 43 43.00 38 146596288 26.4 21.7 21.6 30.3 0.0 35.9 24.6 smartseq 1054361 SRR2088169 SRP060416 SRS980322 SRX1082138 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810604: T75_P1_C2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810604 GSM1810604 T75_P1_C2_ILC3 51584735 1199645 2016-01-28 01:00:06 58525684 51584735 1199645 1 1199645 index:0,count:1199645,average:43,stdev:0 GSM1810604_r1 5.98 6.9 0.24 33591579 41352865 25724548 33148030 123.1 128.86 0 0 0 0 0 0 56.13 74.59 1444076 460803 1444076 460803 61.86 70.93 1444076 507805 1444076 438243 5047845 15.03 6.17 0 16.93 0 1.08 0 0.42 0 0.00 0 30.08 0 820889 0 43 0 41.64 0 1.23 0 0.01 0 1.10 0 0.00 0 239.93 0 0.34 0 73990 0 1199645 0 203078 0 12920 0 4980 0 0 0 360856 0 4 0 0 0 128 0 19932 0 504 0 20568 0 51.50 0 617811 0 4565 22256 4.875355969332 1199645.0 820889.0 73990.0 203078.0 12920.0 4980.0 0.0 360856.0 617811.0 68.4 6.2 16.9 1.1 0.4 0.0 30.1 51.5 43 43 43.00 38 51584735 26.4 21.4 21.8 30.3 0.0 35.3 23.8 smartseq 1054474 SRR2088170 SRP060416 SRS980320 SRX1082139 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810605: T75_P1_C3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810605 GSM1810605 T75_P1_C3_ILC3 148934499 3463593 2016-01-28 01:00:06 162905635 148934499 3463593 1 3463593 index:0,count:3463593,average:43,stdev:0 GSM1810605_r1 2.62 5.19 0.2 105973056 128787009 83219392 106775809 121.53 128.31 0 0 0 0 0 0 59.41 76.58 4592619 1523094 4592619 1523094 64.28 71.93 4592619 1647829 4592619 1430571 13959976 13.17 4.99 0 16.60 0 0.92 0 0.36 0 0.00 0 24.70 0 2563582 0 43 0 41.84 0 1.43 0 0.00 0 1.14 0 0.00 0 389.65 0 0.29 0 172928 0 3463593 0 574784 0 31877 0 12493 0 0 0 855641 0 26 0 0 0 582 0 79227 0 1266 0 81101 0 57.42 0 1988798 0 8351 92219 11.042869117471 3463593.0 2563582.0 172928.0 574784.0 31877.0 12493.0 0.0 855641.0 1988798.0 74.0 5.0 16.6 0.9 0.4 0.0 24.7 57.4 43 43 43.00 38 148934499 26.1 22.3 22.2 29.4 0.0 35.9 24.9 smartseq 1054488 SRR2088171 SRP060416 SRS980321 SRX1082140 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810606: T75_P1_C4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810606 GSM1810606 T75_P1_C4_ILC3 92632922 2154254 2016-01-28 01:00:06 102259079 92632922 2154254 1 2154254 index:0,count:2154254,average:43,stdev:0 GSM1810606_r1 3.18 7.71 0.25 56499139 66816039 44809479 55765888 118.26 124.45 0 0 0 0 0 0 54.98 70.78 2345234 763102 2345234 763102 58.95 68.04 2345234 818188 2345234 733574 10298235 18.23 6.84 0 14.38 0 1.08 0 0.43 0 0.00 0 34.07 0 1387961 0 43 0 41.56 0 1.45 0 0.00 0 1.22 0 0.00 0 267.42 0 0.30 0 147453 0 2154254 0 309872 0 23184 0 9168 0 0 0 733941 0 12 0 0 0 427 0 32259 0 929 0 33627 0 50.04 0 1078089 0 3559 36305 10.200899128969 2154254.0 1387961.0 147453.0 309872.0 23184.0 9168.0 0.0 733941.0 1078089.0 64.4 6.8 14.4 1.1 0.4 0.0 34.1 50.0 43 43 43.00 38 92632922 26.5 21.3 21.2 30.9 0.0 35.8 24.3 smartseq 1054616 SRR2088179 SRP060416 SRS979769 SRX1082148 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810614: T75_P1_D1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810614 GSM1810614 T75_P1_D1_ILC3 71618263 1665541 2016-01-28 01:00:06 79719997 71618263 1665541 1 1665541 index:0,count:1665541,average:43,stdev:0 GSM1810614_r1 1.35 8.13 0.19 39037324 44277367 29483711 35597762 113.42 120.74 0 0 0 0 0 0 53.32 72.36 1802716 516151 1802716 516151 58.12 69.87 1802716 562566 1802716 498400 7143535 18.30 8.10 0 15.29 0 1.15 0 0.29 0 0.00 0 40.45 0 967965 0 43 0 41.33 0 1.28 0 0.00 0 1.13 0 0.00 0 214.14 0 0.33 0 134886 0 1665541 0 254656 0 19125 0 4760 0 0 0 673691 0 8 0 0 0 207 0 20405 0 709 0 21329 0 42.83 0 713309 0 2849 23253 8.161811161811 1665541.0 967965.0 134886.0 254656.0 19125.0 4760.0 0.0 673691.0 713309.0 58.1 8.1 15.3 1.1 0.3 0.0 40.4 42.8 43 43 43.00 38 71618263 26.1 21.5 21.2 31.2 0.0 35.7 23.8 smartseq 1054728 SRR2088180 SRP060416 SRS980312 SRX1082149 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810615: T75_P1_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810615 GSM1810615 T75_P1_D2_NK 47135095 1096165 2016-01-28 01:00:06 52626257 47135095 1096165 1 1096165 index:0,count:1096165,average:43,stdev:0 GSM1810615_r1 2.04 6.81 0.28 29902841 35958479 22867382 29347334 120.25 128.34 0 0 0 0 0 0 55.66 74.07 1349800 407300 1349800 407300 60.96 70.36 1349800 446082 1349800 386897 4695177 15.70 6.45 0 16.59 0 1.09 0 0.28 0 0.00 0 31.88 0 731704 0 43 0 41.59 0 1.39 0 0.00 0 1.21 0 0.00 0 219.23 0 0.32 0 70729 0 1096165 0 181839 0 11947 0 3026 0 0 0 349488 0 10 0 0 0 197 0 18560 0 457 0 19224 0 50.16 0 549865 0 3835 21578 5.626597131682 1096165.0 731704.0 70729.0 181839.0 11947.0 3026.0 0.0 349488.0 549865.0 66.8 6.5 16.6 1.1 0.3 0.0 31.9 50.2 43 43 43.00 38 47135095 26.1 21.8 21.6 30.4 0.0 35.8 24.2 smartseq 1054744 SRR2088181 SRP060416 SRS980311 SRX1082150 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810616: T75_P1_D3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810616 GSM1810616 T75_P1_D3_ILC3 73639220 1712540 2016-01-28 01:00:06 81697735 73639220 1712540 1 1712540 index:0,count:1712540,average:43,stdev:0 GSM1810616_r1 2.71 6.97 0.21 45698784 56179735 35467889 46216502 122.93 130.31 0 0 0 0 0 0 55.71 73.09 1987536 622744 1987536 622744 61.57 69.95 1987536 688316 1987536 596041 7048921 15.42 6.65 0 15.52 0 1.20 0 0.36 0 0.00 0 33.16 0 1117923 0 43 0 41.63 0 1.41 0 0.00 0 1.12 0 0.00 0 205.50 0 0.32 0 113913 0 1712540 0 265869 0 20534 0 6192 0 0 0 567891 0 3 0 0 0 256 0 27360 0 711 0 28330 0 49.75 0 852054 0 4595 32768 7.131229597388 1712540.0 1117923.0 113913.0 265869.0 20534.0 6192.0 0.0 567891.0 852054.0 65.3 6.7 15.5 1.2 0.4 0.0 33.2 49.8 43 43 43.00 38 73639220 26.3 21.8 21.6 30.4 0.0 35.8 24.4 smartseq 1054760 SRR2088182 SRP060416 SRS980310 SRX1082151 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810617: T75_P1_D5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810617 GSM1810617 T75_P1_D5_ILC3 69020117 1605119 2016-01-28 01:00:06 76844216 69020117 1605119 1 1605119 index:0,count:1605119,average:43,stdev:0 GSM1810617_r1 4.1 6.01 0.25 47285492 58806707 37730550 49258132 124.37 130.55 0 0 0 0 0 0 59.68 75.83 1913057 685098 1913057 685098 64.84 72.11 1913057 744308 1913057 651465 6994737 14.79 5.42 0 15.23 0 0.96 0 0.41 0 0.00 0 27.11 0 1147949 0 43 0 41.76 0 1.34 0 0.00 0 1.11 0 0.00 0 240.77 0 0.32 0 87060 0 1605119 0 244516 0 15467 0 6572 0 0 0 435131 0 7 0 0 0 297 0 34624 0 633 0 35561 0 56.28 0 903433 0 6596 39169 5.938295936931 1605119.0 1147949.0 87060.0 244516.0 15467.0 6572.0 0.0 435131.0 903433.0 71.5 5.4 15.2 1.0 0.4 0.0 27.1 56.3 43 43 43.00 38 69020117 26.3 22.1 21.9 29.7 0.0 35.8 24.5 smartseq 1054776 SRR2088183 SRP060416 SRS980307 SRX1082152 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810618: T75_P1_D6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810618 GSM1810618 T75_P1_D6_ILC3 64552503 1501221 2016-01-28 01:00:06 71719798 64552503 1501221 1 1501221 index:0,count:1501221,average:43,stdev:0 GSM1810618_r1 2.62 7.21 0.33 43031759 51807235 34557038 43846633 120.39 126.88 0 0 0 0 0 0 56.89 72.05 1742408 597288 1742408 597288 60.45 68.49 1742408 634671 1742408 567765 6898900 16.03 5.77 0 14.71 0 1.06 0 0.47 0 0.00 0 28.54 0 1049874 0 43 0 41.68 0 1.39 0 0.00 0 1.18 0 0.00 0 360.29 0 0.31 0 86586 0 1501221 0 220850 0 15905 0 7021 0 0 0 428421 0 22 0 0 0 181 0 24462 0 651 0 25316 0 55.22 0 829024 0 3970 27618 6.956675062972 1501221.0 1049874.0 86586.0 220850.0 15905.0 7021.0 0.0 428421.0 829024.0 69.9 5.8 14.7 1.1 0.5 0.0 28.5 55.2 43 43 43.00 38 64552503 26.4 21.7 21.5 30.4 0.0 35.8 24.3 smartseq 1054792 SRR2088184 SRP060416 SRS980308 SRX1082153 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810619: T75_P1_D7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810619 GSM1810619 T75_P1_D7_ILC3 51331207 1193749 2016-01-28 01:00:06 57360783 51331207 1193749 1 1193749 index:0,count:1193749,average:43,stdev:0 GSM1810619_r1 2.91 8.42 0.3 30954562 36976197 23125836 29163128 119.45 126.11 0 0 0 0 0 0 53.54 73.32 1364037 411054 1364037 411054 59.55 69.62 1364037 457174 1364037 390339 5399208 17.44 6.78 0 17.35 0 1.38 0 0.36 0 0.00 0 33.94 0 767742 0 43 0 41.25 0 1.30 0 0.00 0 1.16 0 0.00 0 286.50 0 0.35 0 80928 0 1193749 0 207101 0 16485 0 4318 0 0 0 405204 0 13 0 0 0 185 0 18989 0 512 0 19699 0 46.96 0 560641 0 2726 22912 8.404988994864 1193749.0 767742.0 80928.0 207101.0 16485.0 4318.0 0.0 405204.0 560641.0 64.3 6.8 17.3 1.4 0.4 0.0 33.9 47.0 43 43 43.00 38 51331207 26.7 21.1 20.8 31.3 0.0 35.6 23.7 smartseq 1054808 SRR2088185 SRP060416 SRS980309 SRX1082154 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810620: T75_P1_D8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810620 GSM1810620 T75_P1_D8_ILC3 45268035 1052745 2016-01-28 01:00:06 50636684 45268035 1052745 1 1052745 index:0,count:1052745,average:43,stdev:0 GSM1810620_r1 2.76 7.27 0.21 29056458 35776632 23017023 29617932 123.13 128.68 0 0 0 0 0 0 57.83 74.38 1179598 412225 1179598 412225 62.17 70.34 1179598 443138 1179598 389860 4843276 16.67 6.21 0 15.06 0 1.17 0 0.31 0 0.00 0 30.82 0 712787 0 43 0 41.53 0 1.34 0 0.00 0 1.20 0 0.00 0 236.87 0 0.32 0 65386 0 1052745 0 158557 0 12299 0 3228 0 0 0 324431 0 22 0 0 0 212 0 18053 0 490 0 18777 0 52.65 0 554230 0 2910 20626 7.087972508591 1052745.0 712787.0 65386.0 158557.0 12299.0 3228.0 0.0 324431.0 554230.0 67.7 6.2 15.1 1.2 0.3 0.0 30.8 52.6 43 43 43.00 38 45268035 26.9 21.3 21.2 30.5 0.0 35.7 24.1 smartseq 1054826 SRR2088186 SRP060416 SRS980306 SRX1082155 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810621: T75_P1_E10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810621 GSM1810621 T75_P1_E10_ILC3 37367903 869021 2016-01-28 01:00:06 41926901 37367903 869021 1 869021 index:0,count:869021,average:43,stdev:0 GSM1810621_r1 1.99 8.9 0.25 20877337 24267744 15518959 19177834 116.24 123.58 0 0 0 0 0 0 51.2 70.72 940320 266795 940320 266795 56.6 68.11 940320 294909 940320 256940 3987133 19.10 7.75 0 16.55 0 1.22 0 0.33 0 0.00 0 38.49 0 521046 0 43 0 41.14 0 1.38 0 0.00 0 1.19 0 0.00 0 156.42 0 0.35 0 67317 0 869021 0 143790 0 10601 0 2892 0 0 0 334482 0 0 0 0 0 163 0 12101 0 360 0 12624 0 43.41 0 377256 0 1931 13653 7.070429829104 869021.0 521046.0 67317.0 143790.0 10601.0 2892.0 0.0 334482.0 377256.0 60.0 7.7 16.5 1.2 0.3 0.0 38.5 43.4 43 43 43.00 38 37367903 26.7 21.0 20.7 31.6 0.0 35.5 23.5 smartseq 1054841 SRR2088187 SRP060416 SRS980304 SRX1082156 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810622: T75_P1_E11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810622 GSM1810622 T75_P1_E11_ILC3 84893051 1974257 2016-01-28 01:00:06 94334672 84893051 1974257 1 1974257 index:0,count:1974257,average:43,stdev:0 GSM1810622_r1 2.27 8.49 0.19 52300775 63031208 41060541 51811119 120.52 126.18 0 0 0 0 0 0 53.83 70.17 2095583 696697 2095583 696697 58.36 66.95 2095583 755283 2095583 664756 10123769 19.36 6.57 0 15.26 0 1.22 0 0.34 0 0.00 0 32.89 0 1294228 0 43 0 41.35 0 1.56 0 0.00 0 1.15 0 0.00 0 236.91 0 0.33 0 129730 0 1974257 0 301304 0 24159 0 6626 0 0 0 649244 0 1 0 0 0 317 0 26968 0 977 0 28263 0 50.29 0 992924 0 2247 31498 14.017801513129 1974257.0 1294228.0 129730.0 301304.0 24159.0 6626.0 0.0 649244.0 992924.0 65.6 6.6 15.3 1.2 0.3 0.0 32.9 50.3 43 43 43.00 38 84893051 27.0 20.8 20.5 31.7 0.0 35.6 23.7 smartseq 1054857 SRR2088188 SRP060416 SRS980303 SRX1082157 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810623: T75_P1_E12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810623 GSM1810623 T75_P1_E12_ILC3 87100112 2025584 2016-01-28 01:00:06 96809387 87100112 2025584 1 2025584 index:0,count:2025584,average:43,stdev:0 GSM1810623_r1 4.2 9.53 0.3 51530443 62907896 39184621 50252340 122.08 128.25 0 0 0 0 0 0 53.73 72.45 2167266 688872 2167266 688872 58.88 69.07 2167266 754861 2167266 656743 9357035 18.16 7.03 0 16.35 0 1.18 0 0.37 0 0.00 0 35.16 0 1282037 0 43 0 41.21 0 1.33 0 0.00 0 1.14 0 0.00 0 235.23 0 0.35 0 142390 0 2025584 0 331148 0 23843 0 7566 0 0 0 712138 0 5 0 0 0 378 0 32267 0 997 0 33647 0 46.94 0 950889 0 2249 35719 15.882169853268 2025584.0 1282037.0 142390.0 331148.0 23843.0 7566.0 0.0 712138.0 950889.0 63.3 7.0 16.3 1.2 0.4 0.0 35.2 46.9 43 43 43.00 38 87100112 26.9 20.9 20.6 31.6 0.0 35.6 23.5 smartseq 1054873 SRR2088189 SRP060416 SRS980305 SRX1082158 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810624: T75_P1_E1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810624 GSM1810624 T75_P1_E1_ILC3 142721472 3319104 2016-01-28 01:00:06 157402451 142721472 3319104 1 3319104 index:0,count:3319104,average:43,stdev:0 GSM1810624_r1 3.37 7.49 0.29 83814142 98912705 65021879 80317247 118.01 123.52 0 0 0 0 0 0 52.6 69.17 3548778 1081821 3548778 1081821 57.72 66.04 3548778 1187095 3548778 1032935 15876402 18.94 7.49 0 14.84 0 1.08 0 0.49 0 0.00 0 36.47 0 2056595 0 43 0 41.57 0 1.44 0 0.01 0 1.23 0 0.00 0 234.29 0 0.32 0 248634 0 3319104 0 492509 0 35728 0 16388 0 0 0 1210393 0 15 0 0 0 643 0 49806 0 1394 0 51858 0 47.12 0 1564086 0 4854 57051 11.753399258344 3319104.0 2056595.0 248634.0 492509.0 35728.0 16388.0 0.0 1210393.0 1564086.0 62.0 7.5 14.8 1.1 0.5 0.0 36.5 47.1 43 43 43.00 38 142721472 26.4 21.6 21.4 30.7 0.0 35.7 24.1 smartseq 1054985 SRR2088190 SRP060416 SRS980302 SRX1082159 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810625: T75_P1_E2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810625 GSM1810625 T75_P1_E2_ILC3 64744068 1505676 2016-01-28 01:00:06 73170143 64744068 1505676 1 1505676 index:0,count:1505676,average:43,stdev:0 GSM1810625_r1 3.9 6.54 0.22 44318478 54952175 34304364 44887295 123.99 130.85 0 0 0 0 0 0 57.94 75.97 1892326 624247 1892326 624247 63.76 71.83 1892326 686854 1892326 590224 6462210 14.58 5.41 0 16.98 0 1.07 0 0.57 0 0.00 0 26.81 0 1077318 0 43 0 41.75 0 1.33 0 0.00 0 1.16 0 0.00 0 246.38 0 0.34 0 81446 0 1505676 0 255639 0 16098 0 8652 0 0 0 403608 0 25 0 0 0 358 0 31459 0 520 0 32362 0 54.57 0 821679 0 6513 36922 5.668969752802 1505676.0 1077318.0 81446.0 255639.0 16098.0 8652.0 0.0 403608.0 821679.0 71.6 5.4 17.0 1.1 0.6 0.0 26.8 54.6 43 43 43.00 38 64744068 26.4 21.7 21.8 30.1 0.0 35.4 23.9 smartseq 1055001 SRR2088191 SRP060416 SRS980301 SRX1082160 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810626: T75_P1_E3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810626 GSM1810626 T75_P1_E3_ILC3 117499822 2732554 2016-01-28 01:00:06 129865559 117499822 2732554 1 2732554 index:0,count:2732554,average:43,stdev:0 GSM1810626_r1 2.36 7.51 0.21 71922814 81887413 56020276 66891099 113.85 119.41 0 0 0 0 0 0 49.05 64.3 3092735 866879 3092735 866879 53.78 61.76 3092735 950491 3092735 832684 15173626 21.10 6.79 0 15.33 0 1.14 0 0.45 0 0.00 0 33.74 0 1767236 0 43 0 41.55 0 1.34 0 0.00 0 1.19 0 0.00 0 317.33 0 0.32 0 185450 0 2732554 0 418968 0 31164 0 12322 0 0 0 921832 0 3 0 0 0 439 0 40044 0 1202 0 41688 0 49.34 0 1348268 0 4524 46118 10.194076038904 2732554.0 1767236.0 185450.0 418968.0 31164.0 12322.0 0.0 921832.0 1348268.0 64.7 6.8 15.3 1.1 0.5 0.0 33.7 49.3 43 43 43.00 38 117499822 26.4 21.5 21.2 31.0 0.0 35.7 24.0 smartseq 1055016 SRR2088192 SRP060416 SRS980300 SRX1082161 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810627: T75_P1_E4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810627 GSM1810627 T75_P1_E4_ILC3 66443041 1545187 2016-01-28 01:00:06 75276049 66443041 1545187 1 1545187 index:0,count:1545187,average:43,stdev:0 GSM1810627_r1 2.55 5.47 0.26 47543757 57822360 37228552 48086771 121.62 129.17 0 0 0 0 0 0 55.43 71.77 2063425 638899 2063425 638899 60.77 68.24 2063425 700430 2063425 607483 7320256 15.40 4.94 0 16.99 0 0.98 0 0.39 0 0.00 0 24.03 0 1152661 0 43 0 41.82 0 1.44 0 0.00 0 1.18 0 0.00 0 198.67 0 0.33 0 76380 0 1545187 0 262486 0 15163 0 6052 0 0 0 371311 0 18 0 0 0 242 0 32046 0 582 0 32888 0 57.61 0 890175 0 6073 37169 6.120368845711 1545187.0 1152661.0 76380.0 262486.0 15163.0 6052.0 0.0 371311.0 890175.0 74.6 4.9 17.0 1.0 0.4 0.0 24.0 57.6 43 43 43.00 38 66443041 26.7 21.6 21.7 30.1 0.0 35.3 23.9 smartseq 1055032 SRR2088193 SRP060416 SRS980299 SRX1082162 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810628: T75_P1_E5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810628 GSM1810628 T75_P1_E5_ILC3 103326334 2402938 2016-01-28 01:00:06 114726691 103326334 2402938 1 2402938 index:0,count:2402938,average:43,stdev:0 GSM1810628_r1 3.76 8.03 0.21 63785366 75999704 49555834 61794123 119.15 124.7 0 0 0 0 0 0 54.76 72.03 2671989 859714 2671989 859714 59.43 68.37 2671989 933100 2671989 816065 11791593 18.49 6.64 0 15.67 0 1.07 0 0.36 0 0.00 0 33.23 0 1570068 0 43 0 41.52 0 1.35 0 0.00 0 1.20 0 0.00 0 247.16 0 0.33 0 159665 0 2402938 0 376481 0 25738 0 8732 0 0 0 798400 0 52 0 0 0 297 0 39788 0 978 0 41115 0 49.67 0 1193587 0 4122 44383 10.767345948569 2402938.0 1570068.0 159665.0 376481.0 25738.0 8732.0 0.0 798400.0 1193587.0 65.3 6.6 15.7 1.1 0.4 0.0 33.2 49.7 43 43 43.00 38 103326334 26.5 21.3 21.1 31.2 0.0 35.6 23.9 smartseq 1055048 SRR2088194 SRP060416 SRS980298 SRX1082163 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810629: T75_P1_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810629 GSM1810629 T75_P1_E6_NK 140785182 3274074 2016-01-28 01:00:06 155289886 140785182 3274074 1 3274074 index:0,count:3274074,average:43,stdev:0 GSM1810629_r1 3.29 5.25 0.23 106454978 131142366 81970944 107039156 123.19 130.58 0 0 0 0 0 0 57.8 75.9 4748146 1486123 4748146 1486123 64.79 72.23 4748146 1665798 4748146 1414223 13806553 12.97 4.08 0 18.73 0 1.07 0 0.50 0 0.00 0 19.90 0 2571269 0 43 0 41.86 0 1.45 0 0.00 0 1.21 0 0.00 0 69.33 0 0.31 0 133713 0 3274074 0 613222 0 34916 0 16428 0 0 0 651461 0 34 0 0 0 658 0 76026 0 1204 0 77922 0 59.80 0 1958047 0 7527 89790 11.929055400558 3274074.0 2571269.0 133713.0 613222.0 34916.0 16428.0 0.0 651461.0 1958047.0 78.5 4.1 18.7 1.1 0.5 0.0 19.9 59.8 43 43 43.00 38 140785182 26.6 22.0 21.9 29.5 0.0 35.8 24.6 smartseq 1055065 SRR2088195 SRP060416 SRS980297 SRX1082164 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810630: T75_P1_F10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810630 GSM1810630 T75_P1_F10_ILC3 34025212 791284 2016-01-28 01:00:06 38079119 34025212 791284 1 791284 index:0,count:791284,average:43,stdev:0 GSM1810630_r1 4.41 7.6 0.21 21726540 26830536 16904423 21803566 123.49 128.98 0 0 0 0 0 0 57.89 75.92 886690 309820 886690 309820 63.69 72.82 886690 340843 886690 297180 3354105 15.44 6.13 0 16.06 0 1.23 0 0.41 0 0.00 0 30.72 0 535171 0 43 0 41.42 0 1.29 0 0.00 0 1.17 0 0.00 0 32.01 0 0.33 0 48476 0 791284 0 127068 0 9722 0 3279 0 0 0 243112 0 0 0 0 0 80 0 13541 0 356 0 13977 0 51.57 0 408103 0 2150 15249 7.092558139535 791284.0 535171.0 48476.0 127068.0 9722.0 3279.0 0.0 243112.0 408103.0 67.6 6.1 16.1 1.2 0.4 0.0 30.7 51.6 43 43 43.00 38 34025212 27.0 21.1 20.9 31.0 0.0 35.7 24.0 smartseq 1055082 SRR2088196 SRP060416 SRS980296 SRX1082165 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810631: T75_P1_F11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810631 GSM1810631 T75_P1_F11_ILC3 63348761 1473227 2016-01-28 01:00:06 70362714 63348761 1473227 1 1473227 index:0,count:1473227,average:43,stdev:0 GSM1810631_r1 3.2 7.24 0.29 42953683 53465039 34709019 45032750 124.47 129.74 0 0 0 0 0 0 53.9 67.91 1646138 567488 1646138 567488 57.46 64.01 1646138 605004 1646138 534881 7995827 18.61 5.34 0 14.75 0 1.22 0 0.49 0 0.00 0 26.82 0 1052898 0 43 0 41.53 0 1.39 0 0.00 0 1.26 0 0.00 0 331.48 0 0.32 0 78695 0 1473227 0 217230 0 17991 0 7272 0 0 0 395066 0 0 0 0 0 256 0 21844 0 649 0 22749 0 56.72 0 835668 0 2237 26601 11.891372373715 1473227.0 1052898.0 78695.0 217230.0 17991.0 7272.0 0.0 395066.0 835668.0 71.5 5.3 14.7 1.2 0.5 0.0 26.8 56.7 43 43 43.00 38 63348761 27.0 21.2 21.0 30.8 0.0 35.8 24.2 smartseq 1055098 SRR2088197 SRP060416 SRS980294 SRX1082166 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810632: T75_P1_F12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810632 GSM1810632 T75_P1_F12_ILC3 70300743 1634901 2016-01-28 01:00:06 77946100 70300743 1634901 1 1634901 index:0,count:1634901,average:43,stdev:0 GSM1810632_r1 3.55 7.31 0.18 46328783 57105170 36350695 46646582 123.26 128.32 0 0 0 0 0 0 60.43 78.46 1833680 687863 1833680 687863 64.81 73.66 1833680 737722 1833680 645808 6455522 13.93 5.79 0 16.00 0 1.07 0 0.43 0 0.00 0 28.87 0 1138278 0 43 0 41.46 0 1.26 0 0.00 0 1.10 0 0.00 0 64.68 0 0.32 0 94613 0 1634901 0 261538 0 17557 0 7066 0 0 0 472000 0 3 0 0 0 328 0 31505 0 696 0 32532 0 53.63 0 876740 0 2011 35981 17.892093485828 1634901.0 1138278.0 94613.0 261538.0 17557.0 7066.0 0.0 472000.0 876740.0 69.6 5.8 16.0 1.1 0.4 0.0 28.9 53.6 43 43 43.00 38 70300743 27.1 21.1 20.9 30.8 0.0 35.8 24.2 smartseq 1055114 SRR2088198 SRP060416 SRS980295 SRX1082167 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810633: T75_P1_F1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810633 GSM1810633 T75_P1_F1_ILC3 142158344 3306008 2016-01-28 01:00:06 155914393 142158344 3306008 1 3306008 index:0,count:3306008,average:43,stdev:0 GSM1810633_r1 3.62 5.72 0.25 101933464 126273472 82008173 106285108 123.88 129.6 0 0 0 0 0 0 59.49 74.9 4071515 1468966 4071515 1468966 64.34 71.12 4071515 1588762 4071515 1394909 13687318 13.43 4.82 0 15.36 0 1.02 0 0.53 0 0.00 0 23.76 0 2469177 0 43 0 41.81 0 1.34 0 0.00 0 1.18 0 0.00 0 350.05 0 0.31 0 159514 0 3306008 0 507817 0 33867 0 17493 0 0 0 785471 0 2 0 0 0 788 0 69626 0 1301 0 71717 0 59.33 0 1961360 0 7256 80829 11.139608599779 3306008.0 2469177.0 159514.0 507817.0 33867.0 17493.0 0.0 785471.0 1961360.0 74.7 4.8 15.4 1.0 0.5 0.0 23.8 59.3 43 43 43.00 38 142158344 26.4 22.0 21.9 29.8 0.0 35.9 24.8 smartseq 1055130 SRR2088199 SRP060416 SRS980293 SRX1082168 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810634: T75_P1_F2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810634 GSM1810634 T75_P1_F2_ILC3 42336381 984567 2016-01-28 01:00:06 47913979 42336381 984567 1 984567 index:0,count:984567,average:43,stdev:0 GSM1810634_r1 0.98 7.98 0.26 25126712 28213376 19513172 23153247 112.28 118.65 0 0 0 0 0 0 51.36 67.67 1085846 318435 1085846 318435 55.54 65.1 1085846 344339 1085846 306344 4954195 19.72 7.12 0 15.18 0 1.13 0 0.43 0 0.00 0 35.46 0 620013 0 43 0 41.47 0 1.37 0 0.00 0 1.21 0 0.01 0 221.53 0 0.34 0 70057 0 984567 0 149436 0 11166 0 4255 0 0 0 349133 0 1 0 0 0 161 0 12416 0 428 0 13006 0 47.80 0 470577 0 3257 14645 4.496469143383 984567.0 620013.0 70057.0 149436.0 11166.0 4255.0 0.0 349133.0 470577.0 63.0 7.1 15.2 1.1 0.4 0.0 35.5 47.8 43 43 43.00 38 42336381 26.4 21.2 21.1 31.3 0.0 35.3 23.6 smartseq 1056777 SRR2088200 SRP060416 SRS980292 SRX1082169 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810635: T75_P1_F3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810635 GSM1810635 T75_P1_F3_ILC3 123257221 2866447 2016-01-28 01:00:06 135684709 123257221 2866447 1 2866447 index:0,count:2866447,average:43,stdev:0 GSM1810635_r1 7.36 6.03 0.26 86147193 111035923 67150251 90004786 128.89 134.03 0 0 0 0 0 0 59.44 77.3 3523277 1242395 3523277 1242395 65.68 73.83 3523277 1372827 3523277 1186574 11567599 13.43 5.21 0 16.85 0 0.94 0 0.45 0 0.00 0 25.70 0 2090016 0 43 0 41.78 0 1.27 0 0.01 0 1.16 0 0.00 0 332.88 0 0.30 0 149243 0 2866447 0 482859 0 26819 0 12809 0 0 0 736803 0 13 0 0 0 461 0 56664 0 1183 0 58321 0 56.07 0 1607157 0 6754 63876 9.457506662718 2866447.0 2090016.0 149243.0 482859.0 26819.0 12809.0 0.0 736803.0 1607157.0 72.9 5.2 16.8 0.9 0.4 0.0 25.7 56.1 43 43 43.00 38 123257221 26.4 21.9 21.7 30.0 0.0 35.9 24.8 smartseq 1056794 SRR2088201 SRP060416 SRS980289 SRX1082170 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810636: T75_P1_F4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810636 GSM1810636 T75_P1_F4_ILC3 112105343 2607101 2016-01-28 01:00:06 123447594 112105343 2607101 1 2607101 index:0,count:2607101,average:43,stdev:0 GSM1810636_r1 2.89 3.79 0.18 82349509 94838080 65236111 80672986 115.17 123.66 0 0 0 0 0 0 48.9 62.38 3797582 969988 3797582 969988 52.95 59.65 3797582 1050283 3797582 927629 16311909 19.81 4.67 0 16.44 0 0.99 0 0.51 0 0.00 0 22.42 0 1983521 0 43 0 41.95 0 1.42 0 0.01 0 1.13 0 0.00 0 335.20 0 0.29 0 121753 0 2607101 0 428484 0 25790 0 13210 0 0 0 584580 0 35 0 0 0 381 0 50278 0 802 0 51496 0 59.65 0 1555037 0 6972 56382 8.086919104991 2607101.0 1983521.0 121753.0 428484.0 25790.0 13210.0 0.0 584580.0 1555037.0 76.1 4.7 16.4 1.0 0.5 0.0 22.4 59.6 43 43 43.00 38 112105343 26.7 21.8 21.8 29.6 0.0 36.0 25.1 smartseq 1056809 SRR2088202 SRP060416 SRS980290 SRX1082171 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810637: T75_P1_F5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810637 GSM1810637 T75_P1_F5_ILC3 101521280 2360960 2016-01-28 01:00:06 112325577 101521280 2360960 1 2360960 index:0,count:2360960,average:43,stdev:0 GSM1810637_r1 5.37 6.66 0.35 66963914 84478848 51001693 67752956 126.16 132.84 0 0 0 0 0 0 56.78 75.85 2884086 930097 2884086 930097 63.83 72.55 2884086 1045595 2884086 889723 9336248 13.94 5.76 0 17.44 0 1.33 0 0.55 0 0.00 0 28.74 0 1638060 0 43 0 41.59 0 1.28 0 0.00 0 1.15 0 0.00 0 242.84 0 0.32 0 136090 0 2360960 0 411762 0 31364 0 13096 0 0 0 678440 0 3 0 0 0 357 0 39356 0 991 0 40707 0 51.94 0 1226298 0 4120 46129 11.196359223301 2360960.0 1638060.0 136090.0 411762.0 31364.0 13096.0 0.0 678440.0 1226298.0 69.4 5.8 17.4 1.3 0.6 0.0 28.7 51.9 43 43 43.00 38 101521280 26.4 21.7 21.5 30.4 0.0 35.7 24.3 smartseq 1056825 SRR2088203 SRP060416 SRS980291 SRX1082172 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810638: T75_P1_F6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810638 GSM1810638 T75_P1_F6_ILC3 130879057 3043699 2016-01-28 01:00:06 144217272 130879057 3043699 1 3043699 index:0,count:3043699,average:43,stdev:0 GSM1810638_r1 2.36 7.64 0.33 77399536 89213555 58892037 72186321 115.26 122.57 0 0 0 0 0 0 50.87 68.39 3510087 971252 3510087 971252 56.06 65.64 3510087 1070338 3510087 932220 15023768 19.41 7.13 0 16.07 0 1.18 0 0.41 0 0.00 0 35.68 0 1909357 0 43 0 41.47 0 1.41 0 0.00 0 1.15 0 0.00 0 304.37 0 0.32 0 217125 0 3043699 0 489212 0 36005 0 12360 0 0 0 1085977 0 0 0 0 0 333 0 41123 0 1225 0 42681 0 46.66 0 1420145 0 3859 49476 12.820938066857 3043699.0 1909357.0 217125.0 489212.0 36005.0 12360.0 0.0 1085977.0 1420145.0 62.7 7.1 16.1 1.2 0.4 0.0 35.7 46.7 43 43 43.00 38 130879057 26.4 21.3 21.1 31.2 0.0 35.7 24.0 smartseq 1056840 SRR2088204 SRP060416 SRS980288 SRX1082173 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810639: T75_P1_F7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810639 GSM1810639 T75_P1_F7_ILC3 104047100 2419700 2016-01-28 01:00:06 114965081 104047100 2419700 1 2419700 index:0,count:2419700,average:43,stdev:0 GSM1810639_r1 3.73 8.08 0.29 61737114 73525696 46615958 58671212 119.09 125.86 0 0 0 0 0 0 51.05 69.21 2724379 780806 2724379 780806 57.32 67.01 2724379 876781 2724379 756053 11750815 19.03 6.98 0 16.59 0 1.34 0 0.46 0 0.00 0 34.99 0 1529616 0 43 0 41.32 0 1.55 0 0.01 0 1.22 0 0.01 0 290.36 0 0.32 0 168999 0 2419700 0 401425 0 32334 0 11156 0 0 0 846594 0 1 0 0 0 308 0 30924 0 1039 0 32272 0 46.63 0 1128191 0 2291 36451 15.910519423832 2419700.0 1529616.0 168999.0 401425.0 32334.0 11156.0 0.0 846594.0 1128191.0 63.2 7.0 16.6 1.3 0.5 0.0 35.0 46.6 43 43 43.00 38 104047100 27.0 20.9 20.7 31.5 0.0 35.7 23.9 smartseq 1056857 SRR2088205 SRP060416 SRS979770 SRX1082174 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810640: T75_P1_F8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810640 GSM1810640 T75_P1_F8_ILC3 70760886 1645602 2016-01-28 01:00:06 78613486 70760886 1645602 1 1645602 index:0,count:1645602,average:43,stdev:0 GSM1810640_r1 3.3 6.53 0.23 49797066 61609635 40102480 51432475 123.72 128.25 0 0 0 0 0 0 57.2 72.13 1923866 694660 1923866 694660 62.13 68.77 1923866 754484 1923866 662298 7664839 15.39 4.95 0 15.28 0 1.23 0 0.45 0 0.00 0 24.51 0 1214441 0 43 0 41.64 0 1.22 0 0.00 0 1.18 0 0.01 0 311.80 0 0.32 0 81385 0 1645602 0 251393 0 20291 0 7451 0 0 0 403419 0 3 0 0 0 306 0 32648 0 720 0 33677 0 58.52 0 963048 0 2634 38354 14.561123766135 1645602.0 1214441.0 81385.0 251393.0 20291.0 7451.0 0.0 403419.0 963048.0 73.8 4.9 15.3 1.2 0.5 0.0 24.5 58.5 43 43 43.00 38 70760886 27.0 21.4 21.3 30.2 0.0 35.8 24.5 smartseq 1056872 SRR2088206 SRP060416 SRS980286 SRX1082175 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810641: T75_P1_G10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810641 GSM1810641 T75_P1_G10_ILC3 62767745 1459715 2016-01-28 01:00:06 69668094 62767745 1459715 1 1459715 index:0,count:1459715,average:43,stdev:0 GSM1810641_r1 1.56 9.49 0.4 33753490 39263241 25581518 31392801 116.32 122.72 0 0 0 0 0 0 53.84 72.99 1473950 452944 1473950 452944 58.14 69.02 1473950 489094 1473950 428341 6136102 18.18 8.04 0 15.11 0 1.12 0 0.28 0 0.00 0 40.98 0 841212 0 43 0 41.22 0 1.34 0 0.00 0 1.15 0 0.00 0 262.75 0 0.33 0 117422 0 1459715 0 220620 0 16325 0 4026 0 0 0 598152 0 17 0 0 0 243 0 19343 0 632 0 20235 0 42.51 0 620592 0 1873 22394 11.956219967966 1459715.0 841212.0 117422.0 220620.0 16325.0 4026.0 0.0 598152.0 620592.0 57.6 8.0 15.1 1.1 0.3 0.0 41.0 42.5 43 43 43.00 38 62767745 26.5 21.1 21.0 31.3 0.0 35.7 24.0 smartseq 1056888 SRR2088207 SRP060416 SRS980285 SRX1082176 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810642: T75_P1_G1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810642 GSM1810642 T75_P1_G1_ILC3 139842321 3252147 2016-01-28 01:00:06 152902878 139842321 3252147 1 3252147 index:0,count:3252147,average:43,stdev:0 GSM1810642_r1 2.59 6.92 0.24 88101593 106505028 68924091 88571460 120.89 128.51 0 0 0 0 0 0 57.35 74.66 3817649 1235764 3817649 1235764 61.84 71.14 3817649 1332533 3817649 1177473 13427754 15.24 6.49 0 15.36 0 1.01 0 0.38 0 0.00 0 32.35 0 2154650 0 43 0 41.64 0 1.56 0 0.01 0 1.16 0 0.00 0 292.69 0 0.30 0 211155 0 3252147 0 499561 0 32815 0 12500 0 0 0 1052182 0 38 0 0 0 646 0 53066 0 1207 0 54957 0 50.89 0 1655089 0 4443 61791 13.907494935854 3252147.0 2154650.0 211155.0 499561.0 32815.0 12500.0 0.0 1052182.0 1655089.0 66.3 6.5 15.4 1.0 0.4 0.0 32.4 50.9 43 43 43.00 38 139842321 26.3 21.6 21.5 30.6 0.0 35.9 24.5 smartseq 1056905 SRR2088208 SRP060416 SRS980287 SRX1082177 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810643: T75_P1_G3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810643 GSM1810643 T75_P1_G3_ILC3 112799965 2623255 2016-01-28 01:00:06 123538347 112799965 2623255 1 2623255 index:0,count:2623255,average:43,stdev:0 GSM1810643_r1 4.17 6.66 0.2 73553593 90047512 56964929 73215865 122.42 128.53 0 0 0 0 0 0 57.51 75.49 3143139 1032393 3143139 1032393 63.15 71.89 3143139 1133669 3143139 983133 10849097 14.75 6.10 0 16.31 0 1.00 0 0.33 0 0.00 0 30.23 0 1795280 0 43 0 41.66 0 1.31 0 0.00 0 1.09 0 0.00 0 277.76 0 0.29 0 159914 0 2623255 0 427758 0 26102 0 8739 0 0 0 793134 0 14 0 0 0 537 0 45933 0 957 0 47441 0 52.13 0 1367522 0 5715 53335 9.332458442695 2623255.0 1795280.0 159914.0 427758.0 26102.0 8739.0 0.0 793134.0 1367522.0 68.4 6.1 16.3 1.0 0.3 0.0 30.2 52.1 43 43 43.00 38 112799965 26.2 21.8 21.6 30.4 0.0 36.0 24.7 smartseq 1056921 SRR2088209 SRP060416 SRS980284 SRX1082178 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810644: T75_P1_G5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810644 GSM1810644 T75_P1_G5_ILC3 125232383 2912381 2016-01-28 01:00:06 137526247 125232383 2912381 1 2912381 index:0,count:2912381,average:43,stdev:0 GSM1810644_r1 2.2 7.73 0.28 73367131 86987328 56471939 70966773 118.56 125.67 0 0 0 0 0 0 55.33 73.51 3228284 999975 3228284 999975 60.25 70.33 3228284 1088796 3228284 956751 12412444 16.92 7.23 0 15.34 0 1.07 0 0.38 0 0.00 0 36.49 0 1807131 0 43 0 41.51 0 1.36 0 0.00 0 1.15 0 0.00 0 327.64 0 0.31 0 210591 0 2912381 0 446767 0 31266 0 11134 0 0 0 1062850 0 15 0 0 0 572 0 43069 0 1202 0 44858 0 46.71 0 1360364 0 3681 49825 13.535723988047 2912381.0 1807131.0 210591.0 446767.0 31266.0 11134.0 0.0 1062850.0 1360364.0 62.0 7.2 15.3 1.1 0.4 0.0 36.5 46.7 43 43 43.00 38 125232383 26.2 21.6 21.4 30.8 0.0 35.8 24.3 smartseq 1057032 SRR2088210 SRP060416 SRS980283 SRX1082179 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810645: T75_P1_G6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810645 GSM1810645 T75_P1_G6_ILC3 141148403 3282521 2016-01-28 01:00:06 155350822 141148403 3282521 1 3282521 index:0,count:3282521,average:43,stdev:0 GSM1810645_r1 4.54 6.0 0.2 98990018 124461094 78674764 103317919 125.73 131.32 0 0 0 0 0 0 58.63 74.79 3995116 1407384 3995116 1407384 64.17 71.37 3995116 1540241 3995116 1343033 14255913 14.40 5.22 0 15.80 0 1.01 0 0.43 0 0.00 0 25.43 0 2400354 0 43 0 41.81 0 1.42 0 0.00 0 1.16 0 0.00 0 77.24 0 0.30 0 171264 0 3282521 0 518685 0 33227 0 14138 0 0 0 834802 0 86 0 0 0 610 0 62352 0 1219 0 64267 0 57.32 0 1881669 0 5914 73279 12.390767669936 3282521.0 2400354.0 171264.0 518685.0 33227.0 14138.0 0.0 834802.0 1881669.0 73.1 5.2 15.8 1.0 0.4 0.0 25.4 57.3 43 43 43.00 38 141148403 26.4 21.9 21.9 29.9 0.0 35.8 24.7 smartseq 1057049 SRR2088211 SRP060416 SRS980282 SRX1082180 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810646: T75_P1_G7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810646 GSM1810646 T75_P1_G7_ILC3 87743607 2040549 2016-01-28 01:00:06 97569653 87743607 2040549 1 2040549 index:0,count:2040549,average:43,stdev:0 GSM1810646_r1 5.24 7.57 0.27 57263384 70915866 44831979 57800098 123.84 128.93 0 0 0 0 0 0 56.77 73.95 2270901 799010 2270901 799010 62.24 70.53 2270901 875916 2270901 762125 8791023 15.35 5.93 0 16.02 0 1.12 0 0.35 0 0.00 0 29.56 0 1407428 0 43 0 41.49 0 1.36 0 0.00 0 1.17 0 0.00 0 293.84 0 0.32 0 121071 0 2040549 0 326900 0 22850 0 7090 0 0 0 603181 0 2 0 0 0 270 0 33651 0 913 0 34836 0 52.95 0 1080528 0 2706 39048 14.430155210643 2040549.0 1407428.0 121071.0 326900.0 22850.0 7090.0 0.0 603181.0 1080528.0 69.0 5.9 16.0 1.1 0.3 0.0 29.6 53.0 43 43 43.00 38 87743607 27.1 21.0 20.9 30.9 0.0 35.6 24.1 smartseq 1057066 SRR2088212 SRP060416 SRS980281 SRX1082181 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810647: T75_P1_G8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810647 GSM1810647 T75_P1_G8_ILC3 87088545 2025315 2016-01-28 01:00:06 96125962 87088545 2025315 1 2025315 index:0,count:2025315,average:43,stdev:0 GSM1810647_r1 3.22 7.46 0.26 54854699 65888916 42450779 53380326 120.12 125.75 0 0 0 0 0 0 56.91 74.99 2334475 765995 2334475 765995 62.89 71.74 2334475 846582 2334475 732726 8349269 15.22 6.37 0 16.03 0 1.04 0 0.37 0 0.00 0 32.12 0 1346038 0 43 0 41.56 0 1.25 0 0.00 0 1.21 0 0.00 0 182.28 0 0.31 0 128994 0 2025315 0 324633 0 21143 0 7524 0 0 0 650610 0 21 0 0 0 331 0 32229 0 831 0 33412 0 50.43 0 1021405 0 2408 37484 15.566445182724 2025315.0 1346038.0 128994.0 324633.0 21143.0 7524.0 0.0 650610.0 1021405.0 66.5 6.4 16.0 1.0 0.4 0.0 32.1 50.4 43 43 43.00 38 87088545 26.8 21.3 21.3 30.6 0.0 35.8 24.4 smartseq 1057081 SRR2088213 SRP060416 SRS980280 SRX1082182 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810648: T75_P1_H11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810648 GSM1810648 T75_P1_H11_ILC3 94002472 2186104 2016-01-28 01:00:06 105091502 94002472 2186104 1 2186104 index:0,count:2186104,average:43,stdev:0 GSM1810648_r1 2.65 8.07 0.27 58718670 70414267 45675185 57560389 119.92 126.02 0 0 0 0 0 0 54.97 72.16 2428070 795964 2428070 795964 59.84 69.06 2428070 866604 2428070 761770 10228903 17.42 6.42 0 15.78 0 1.21 0 0.51 0 0.00 0 32.03 0 1448109 0 43 0 41.41 0 1.34 0 0.00 0 1.15 0 0.01 0 231.47 0 0.34 0 140283 0 2186104 0 345001 0 26539 0 11159 0 0 0 700297 0 10 0 0 0 333 0 36522 0 995 0 37860 0 50.46 0 1103108 0 3347 41387 12.365401852405 2186104.0 1448109.0 140283.0 345001.0 26539.0 11159.0 0.0 700297.0 1103108.0 66.2 6.4 15.8 1.2 0.5 0.0 32.0 50.5 43 43 43.00 38 94002472 26.8 21.1 20.8 31.4 0.0 35.4 23.6 smartseq 1057097 SRR2088214 SRP060416 SRS980279 SRX1082183 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810649: T75_P1_H2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810649 GSM1810649 T75_P1_H2_ILC3 30890727 718389 2016-01-28 01:00:06 35863927 30890727 718389 1 718389 index:0,count:718389,average:43,stdev:0 GSM1810649_r1 3.44 6.79 0.17 21563443 26247404 17105810 21846393 121.72 127.71 0 0 0 0 0 0 54.62 69.98 881491 287028 881491 287028 59.29 66.53 881491 311554 881491 272876 3705899 17.19 5.22 0 16.06 0 1.02 0 0.42 0 0.00 0 25.41 0 525518 0 43 0 41.71 0 1.40 0 0.00 0 1.15 0 0.00 0 143.68 0 0.40 0 37534 0 718389 0 115366 0 7355 0 2995 0 0 0 182521 0 5 0 0 0 153 0 13109 0 252 0 13519 0 57.09 0 410152 0 5010 14598 2.913772455090 718389.0 525518.0 37534.0 115366.0 7355.0 2995.0 0.0 182521.0 410152.0 73.2 5.2 16.1 1.0 0.4 0.0 25.4 57.1 43 43 43.00 38 30890727 26.6 20.9 21.9 30.6 0.0 34.6 22.9 smartseq 1057114 SRR2088215 SRP060416 SRS980278 SRX1082184 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810650: T75_P1_H3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810650 GSM1810650 T75_P1_H3_ILC3 102966811 2394577 2016-01-28 01:00:06 115078232 102966811 2394577 1 2394577 index:0,count:2394577,average:43,stdev:0 GSM1810650_r1 3.44 6.93 0.28 65667832 79049899 50002020 63613982 120.38 127.22 0 0 0 0 0 0 55.05 73.64 2920358 885816 2920358 885816 61.2 70.29 2920358 984651 2920358 845457 10550575 16.07 6.33 0 16.96 0 1.08 0 0.33 0 0.00 0 31.40 0 1608973 0 43 0 41.57 0 1.34 0 0.00 0 1.20 0 0.00 0 253.54 0 0.33 0 151629 0 2394577 0 406137 0 25825 0 7885 0 0 0 751894 0 5 0 0 0 319 0 39475 0 978 0 40777 0 50.23 0 1202836 0 5250 45731 8.710666666667 2394577.0 1608973.0 151629.0 406137.0 25825.0 7885.0 0.0 751894.0 1202836.0 67.2 6.3 17.0 1.1 0.3 0.0 31.4 50.2 43 43 43.00 38 102966811 26.2 21.6 21.4 30.8 0.0 35.5 23.8 smartseq 1057130 SRR2088216 SRP060416 SRS980277 SRX1082185 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810651: T75_P1_H4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810651 GSM1810651 T75_P1_H4_ILC3 73873957 1717999 2016-01-28 01:00:06 82727968 73873957 1717999 1 1717999 index:0,count:1717999,average:43,stdev:0 GSM1810651_r1 2.34 8.97 0.3 41683597 47895729 32595494 39415464 114.9 120.92 0 0 0 0 0 0 52.48 68.83 1733237 542596 1733237 542596 55.94 65.84 1733237 578320 1733237 518997 8197618 19.67 7.70 0 14.30 0 1.15 0 0.34 0 0.00 0 38.33 0 1033888 0 43 0 41.35 0 1.33 0 0.00 0 1.12 0 0.00 0 193.27 0 0.34 0 132268 0 1717999 0 245628 0 19769 0 5917 0 0 0 658425 0 3 0 0 0 241 0 21335 0 818 0 22397 0 45.88 0 788260 0 3224 24010 7.447270471464 1717999.0 1033888.0 132268.0 245628.0 19769.0 5917.0 0.0 658425.0 788260.0 60.2 7.7 14.3 1.2 0.3 0.0 38.3 45.9 43 43 43.00 38 73873957 26.5 21.2 20.9 31.4 0.0 35.4 23.5 smartseq 1057146 SRR2088217 SRP060416 SRS980275 SRX1082186 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810652: T75_P1_H5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810652 GSM1810652 T75_P1_H5_ILC3 163609625 3804875 2016-01-28 01:00:06 180692782 163609625 3804875 1 3804875 index:0,count:3804875,average:43,stdev:0 GSM1810652_r1 4.42 5.92 0.2 116905477 146918687 92308163 120993152 125.67 131.08 0 0 0 0 0 0 60.25 77.3 4759054 1706126 4759054 1706126 66.76 73.78 4759054 1890352 4759054 1628516 15175736 12.98 4.87 0 16.41 0 1.00 0 0.46 0 0.00 0 24.13 0 2831529 0 43 0 41.82 0 1.54 0 0.01 0 1.23 0 0.00 0 351.22 0 0.32 0 185144 0 3804875 0 624261 0 37976 0 17339 0 0 0 918031 0 34 0 0 0 706 0 80329 0 1440 0 82509 0 58.01 0 2207268 0 6774 93253 13.766312370830 3804875.0 2831529.0 185144.0 624261.0 37976.0 17339.0 0.0 918031.0 2207268.0 74.4 4.9 16.4 1.0 0.5 0.0 24.1 58.0 43 43 43.00 38 163609625 26.5 21.9 21.8 29.7 0.0 35.7 24.4 smartseq 1057161 SRR2088218 SRP060416 SRS980276 SRX1082187 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810653: T75_P1_H8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810653 GSM1810653 T75_P1_H8_ILC3 80033363 1861241 2016-01-28 01:00:06 89877917 80033363 1861241 1 1861241 index:0,count:1861241,average:43,stdev:0 GSM1810653_r1 1.87 7.79 0.28 45616677 53099128 36415692 44707529 116.4 122.77 0 0 0 0 0 0 54.14 69.31 1888668 607806 1888668 607806 57.2 66.2 1888668 642137 1888668 580518 8546200 18.73 8.06 0 13.20 0 1.04 0 0.34 0 0.00 0 38.31 0 1122596 0 43 0 41.53 0 1.59 0 0.01 0 1.13 0 0.00 0 239.30 0 0.36 0 150033 0 1861241 0 245670 0 19370 0 6293 0 0 0 712982 0 2 0 0 0 207 0 21809 0 756 0 22774 0 47.12 0 876926 0 1840 24927 13.547282608696 1861241.0 1122596.0 150033.0 245670.0 19370.0 6293.0 0.0 712982.0 876926.0 60.3 8.1 13.2 1.0 0.3 0.0 38.3 47.1 43 43 43.00 38 80033363 27.1 21.2 21.2 30.5 0.0 35.4 23.8 smartseq 1057176 SRR2088219 SRP060416 SRS980274 SRX1082188 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810654: T75_P2_A1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810654 GSM1810654 T75_P2_A1_ILC3 38336177 891539 2016-01-28 01:00:06 44691968 38336177 891539 1 891539 index:0,count:891539,average:43,stdev:0 GSM1810654_r1 3.44 8.6 0.19 24618113 29524510 18656100 23662571 119.93 126.84 0 0 0 0 0 0 55.77 75.16 1070415 337980 1070415 337980 61.67 71.69 1070415 373715 1070415 322372 3742852 15.20 6.46 0 17.53 0 1.09 0 0.31 0 0.00 0 30.63 0 605998 0 43 0 41.49 0 1.42 0 0.00 0 1.16 0 0.00 0 213.97 0 0.40 0 57559 0 891539 0 156318 0 9694 0 2771 0 0 0 273076 0 0 0 0 0 151 0 15083 0 306 0 15540 0 50.44 0 449680 0 4168 17346 4.161708253359 891539.0 605998.0 57559.0 156318.0 9694.0 2771.0 0.0 273076.0 449680.0 68.0 6.5 17.5 1.1 0.3 0.0 30.6 50.4 43 43 43.00 38 38336177 26.1 20.7 21.5 31.6 0.0 34.4 22.4 smartseq 1057288 SRR2088220 SRP060416 SRS980273 SRX1082189 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810655: T75_P2_A3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810655 GSM1810655 T75_P2_A3_ILC3 97153985 2259395 2016-01-28 01:00:06 109288609 97153985 2259395 1 2259395 index:0,count:2259395,average:43,stdev:0 GSM1810655_r1 4.32 7.37 0.29 60381127 73164950 46964582 59559676 121.17 126.82 0 0 0 0 0 0 55.16 72.29 2523831 813915 2523831 813915 61.07 69.18 2523831 901141 2523831 778929 10062411 16.66 6.73 0 15.48 0 0.98 0 0.32 0 0.00 0 33.39 0 1475573 0 43 0 41.71 0 1.28 0 0.00 0 1.11 0 0.00 0 290.49 0 0.32 0 152134 0 2259395 0 349670 0 22071 0 7275 0 0 0 754476 0 15 0 0 0 337 0 36251 0 715 0 37318 0 49.83 0 1125903 0 4911 42310 8.615353288536 2259395.0 1475573.0 152134.0 349670.0 22071.0 7275.0 0.0 754476.0 1125903.0 65.3 6.7 15.5 1.0 0.3 0.0 33.4 49.8 43 43 43.00 38 97153985 26.2 21.5 21.5 30.7 0.0 35.4 23.6 smartseq 1057386 SRR2088226 SRP060416 SRS979771 SRX1082195 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810661: T75_P2_B1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810661 GSM1810661 T75_P2_B1_ILC3 140591897 3269579 2016-01-28 01:00:06 156579755 140591897 3269579 1 3269579 index:0,count:3269579,average:43,stdev:0 GSM1810661_r1 3.07 6.59 0.2 91005104 110327226 70432571 89768663 121.23 127.45 0 0 0 0 0 0 56.82 74.59 3930290 1258076 3930290 1258076 62.5 70.97 3930290 1383810 3930290 1196951 13705455 15.06 6.24 0 16.13 0 0.96 0 0.41 0 0.00 0 30.92 0 2213958 0 43 0 41.76 0 1.56 0 0.00 0 1.19 0 0.00 0 287.08 0 0.32 0 203920 0 3269579 0 527399 0 31349 0 13320 0 0 0 1010952 0 15 0 0 0 611 0 60422 0 1101 0 62149 0 51.58 0 1686559 0 5693 71253 12.515896715264 3269579.0 2213958.0 203920.0 527399.0 31349.0 13320.0 0.0 1010952.0 1686559.0 67.7 6.2 16.1 1.0 0.4 0.0 30.9 51.6 43 43 43.00 38 140591897 26.1 22.0 22.0 29.8 0.0 35.5 23.9 smartseq 1057401 SRR2088227 SRP060416 SRS980267 SRX1082196 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810662: T75_P2_B3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810662 GSM1810662 T75_P2_B3_ILC3 132824377 3088939 2016-01-28 01:00:06 148109268 132824377 3088939 1 3088939 index:0,count:3088939,average:43,stdev:0 GSM1810662_r1 2.94 6.28 0.23 90313880 111909244 70708050 92195106 123.91 130.39 0 0 0 0 0 0 59.67 77.24 3825998 1303990 3825998 1303990 65.77 73.47 3825998 1437177 3825998 1240267 11383237 12.60 5.63 0 16.09 0 0.92 0 0.35 0 0.00 0 27.98 0 2185298 0 43 0 41.88 0 1.38 0 0.00 0 1.16 0 0.00 0 258.61 0 0.31 0 173889 0 3088939 0 497143 0 28515 0 10832 0 0 0 864294 0 31 0 0 0 606 0 64991 0 911 0 66539 0 54.65 0 1688155 0 6549 76797 11.726523133303 3088939.0 2185298.0 173889.0 497143.0 28515.0 10832.0 0.0 864294.0 1688155.0 70.7 5.6 16.1 0.9 0.4 0.0 28.0 54.7 43 43 43.00 38 132824377 26.2 22.1 22.1 29.6 0.0 35.6 24.1 smartseq 1057417 SRR2088228 SRP060416 SRS980265 SRX1082197 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810663: T75_P2_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810663 GSM1810663 T75_P2_B4_NK 99498302 2313914 2016-01-28 01:00:06 111323790 99498302 2313914 1 2313914 index:0,count:2313914,average:43,stdev:0 GSM1810663_r1 1.9 6.48 0.21 63130891 74792045 49436065 62449096 118.47 126.32 0 0 0 0 0 0 53.7 69.71 2789901 825300 2789901 825300 57.96 66.36 2789901 890902 2789901 785590 11004870 17.43 6.47 0 15.26 0 1.02 0 0.47 0 0.00 0 32.09 0 1536972 0 43 0 41.76 0 1.42 0 0.01 0 1.21 0 0.00 0 231.39 0 0.32 0 149817 0 2313914 0 353147 0 23500 0 10921 0 0 0 742521 0 4 0 0 0 334 0 35908 0 755 0 37001 0 51.16 0 1183825 0 5100 42626 8.358039215686 2313914.0 1536972.0 149817.0 353147.0 23500.0 10921.0 0.0 742521.0 1183825.0 66.4 6.5 15.3 1.0 0.5 0.0 32.1 51.2 43 43 43.00 38 99498302 26.1 22.0 22.0 29.9 0.0 35.5 23.9 smartseq 1057432 SRR2088229 SRP060416 SRS980264 SRX1082198 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810664: T75_P2_B5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810664 GSM1810664 T75_P2_B5_ILC3 137577468 3199476 2016-01-28 01:00:06 153175391 137577468 3199476 1 3199476 index:0,count:3199476,average:43,stdev:0 GSM1810664_r1 5.6 4.7 0.18 100653133 125925297 78052938 102315975 125.11 131.09 0 0 0 0 0 0 58.3 75.99 4303048 1415115 4303048 1415115 64.49 71.7 4303048 1565364 4303048 1335189 13524170 13.44 4.68 0 17.66 0 0.92 0 0.52 0 0.00 0 22.70 0 2427289 0 43 0 41.92 0 1.25 0 0.00 0 1.16 0 0.01 0 426.60 0 0.32 0 149616 0 3199476 0 565140 0 29364 0 16698 0 0 0 726125 0 30 0 0 0 726 0 65596 0 1035 0 67387 0 58.20 0 1862149 0 7264 76922 10.589482378855 3199476.0 2427289.0 149616.0 565140.0 29364.0 16698.0 0.0 726125.0 1862149.0 75.9 4.7 17.7 0.9 0.5 0.0 22.7 58.2 43 43 43.00 38 137577468 26.6 22.1 22.1 29.3 0.0 35.6 24.2 smartseq 1057560 SRR2088231 SRP060416 SRS980266 SRX1082200 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810666: T75_P2_B7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810666 GSM1810666 T75_P2_B7_ILC3 146020217 3395819 2016-01-28 01:00:06 162386660 146020217 3395819 1 3395819 index:0,count:3395819,average:43,stdev:0 GSM1810666_r1 2.98 6.47 0.24 96624599 116533069 76861512 96768887 120.6 125.9 0 0 0 0 0 0 55.39 70.75 3851171 1301964 3851171 1301964 59.95 66.79 3851171 1409136 3851171 1229032 16879990 17.47 5.83 0 15.03 0 1.15 0 0.60 0 0.00 0 29.03 0 2350472 0 43 0 41.77 0 1.42 0 0.00 0 1.24 0 0.00 0 80.43 0 0.33 0 197838 0 3395819 0 510340 0 39175 0 20456 0 0 0 985716 0 55 0 0 0 610 0 61579 0 1185 0 63429 0 54.19 0 1840132 0 4892 71763 14.669460343418 3395819.0 2350472.0 197838.0 510340.0 39175.0 20456.0 0.0 985716.0 1840132.0 69.2 5.8 15.0 1.2 0.6 0.0 29.0 54.2 43 43 43.00 38 146020217 26.7 21.6 21.6 30.1 0.0 35.5 23.9 smartseq 1057576 SRR2088232 SRP060416 SRS980262 SRX1082201 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810667: T75_P2_B8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810667 GSM1810667 T75_P2_B8_ILC3 30558165 710655 2016-01-28 01:00:06 35564725 30558165 710655 1 710655 index:0,count:710655,average:43,stdev:0 GSM1810667_r1 3.57 5.28 0.26 24965426 31347007 20525527 26518564 125.56 129.2 0 0 0 0 0 0 59.62 73.27 924955 358956 924955 358956 65.02 69.84 924955 391426 924955 342157 3347591 13.41 2.92 0 15.77 0 1.08 0 0.64 0 0.00 0 13.56 0 602042 0 43 0 41.89 0 1.50 0 0.00 0 1.14 0 0.00 0 170.56 0 0.38 0 20721 0 710655 0 112103 0 7708 0 4525 0 0 0 96380 0 0 0 0 0 108 0 17736 0 246 0 18090 0 68.94 0 489939 0 4106 19700 4.797856794934 710655.0 602042.0 20721.0 112103.0 7708.0 4525.0 0.0 96380.0 489939.0 84.7 2.9 15.8 1.1 0.6 0.0 13.6 68.9 43 43 43.00 38 30558165 28.0 20.7 21.7 29.6 0.0 34.7 23.3 smartseq 1057592 SRR2088233 SRP060416 SRS980261 SRX1082202 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810668: T75_P2_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810668 GSM1810668 T75_P2_B9_NK 26135486 607802 2016-01-28 01:00:06 29572348 26135486 607802 1 607802 index:0,count:607802,average:43,stdev:0 GSM1810668_r1 2.54 7.43 0.18 15858536 19042466 12067443 15434489 120.08 127.9 0 0 0 0 0 0 53.98 72.39 706389 210133 706389 210133 60.28 69.97 706389 234635 706389 203105 2728480 17.21 6.89 0 16.29 0 1.15 0 0.48 0 0.00 0 34.32 0 389257 0 43 0 41.57 0 1.31 0 0.00 0 1.12 0 0.00 0 136.76 0 0.34 0 41861 0 607802 0 98995 0 6980 0 2943 0 0 0 208622 0 5 0 0 0 141 0 9076 0 228 0 9450 0 47.76 0 290262 0 2228 10597 4.756283662478 607802.0 389257.0 41861.0 98995.0 6980.0 2943.0 0.0 208622.0 290262.0 64.0 6.9 16.3 1.1 0.5 0.0 34.3 47.8 43 43 43.00 38 26135486 26.7 21.4 21.4 30.5 0.0 35.4 23.6 smartseq 1057608 SRR2088234 SRP060416 SRS980260 SRX1082203 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810669: T75_P2_C10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810669 GSM1810669 T75_P2_C10_ILC3 31749953 738371 2016-01-28 01:00:06 36455921 31749953 738371 1 738371 index:0,count:738371,average:43,stdev:0 GSM1810669_r1 1.76 8.67 0.35 18778400 22122311 14397294 17910752 117.81 124.4 0 0 0 0 0 0 53.53 71.48 806771 248754 806771 248754 58.29 68.27 806771 270893 806771 237562 3354706 17.86 7.22 0 15.81 0 1.19 0 0.42 0 0.00 0 35.46 0 464707 0 43 0 41.37 0 1.30 0 0.00 0 1.15 0 0.00 0 166.13 0 0.37 0 53281 0 738371 0 116714 0 8770 0 3096 0 0 0 261798 0 12 0 0 0 108 0 10117 0 241 0 10478 0 47.13 0 347993 0 2256 11634 5.156914893617 738371.0 464707.0 53281.0 116714.0 8770.0 3096.0 0.0 261798.0 347993.0 62.9 7.2 15.8 1.2 0.4 0.0 35.5 47.1 43 43 43.00 38 31749953 26.9 20.8 21.0 31.2 0.0 35.0 23.0 smartseq 1057624 SRR2088235 SRP060416 SRS980258 SRX1082204 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810670: T75_P2_C11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810670 GSM1810670 T75_P2_C11_ILC3 112169499 2608593 2016-01-28 01:00:06 125316666 112169499 2608593 1 2608593 index:0,count:2608593,average:43,stdev:0 GSM1810670_r1 3.51 7.36 0.24 70743385 85477769 55449646 69571930 120.83 125.47 0 0 0 0 0 0 58.35 75.9 2876672 1012495 2876672 1012495 63.87 72.52 2876672 1108266 2876672 967443 11018066 15.57 6.47 0 15.37 0 1.06 0 0.35 0 0.00 0 32.07 0 1735136 0 43 0 41.56 0 1.41 0 0.00 0 1.23 0 0.00 0 335.39 0 0.33 0 168899 0 2608593 0 401071 0 27772 0 9175 0 0 0 836510 0 4 0 0 0 429 0 42177 0 939 0 43549 0 51.14 0 1334065 0 3171 48015 15.141911069063 2608593.0 1735136.0 168899.0 401071.0 27772.0 9175.0 0.0 836510.0 1334065.0 66.5 6.5 15.4 1.1 0.4 0.0 32.1 51.1 43 43 43.00 38 112169499 26.8 21.3 21.3 30.6 0.0 35.5 23.6 smartseq 1057656 SRR2088237 SRP060416 SRS980257 SRX1082206 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810672: T75_P2_C2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810672 GSM1810672 T75_P2_C2_ILC3 47702652 1109364 2016-01-28 01:00:06 54761892 47702652 1109364 1 1109364 index:0,count:1109364,average:43,stdev:0 GSM1810672_r1 2.27 6.58 0.24 33613111 39807144 27150790 33429136 118.43 123.12 0 0 0 0 0 0 52.61 66.04 1323482 428150 1323482 428150 56.62 62.39 1323482 460787 1323482 404489 6109620 18.18 5.11 0 14.92 0 1.00 0 0.52 0 0.00 0 25.13 0 813851 0 43 0 41.88 0 1.39 0 0.00 0 1.15 0 0.00 0 249.61 0 0.35 0 56672 0 1109364 0 165562 0 11055 0 5718 0 0 0 278740 0 6 0 0 0 133 0 19507 0 374 0 20020 0 58.44 0 648289 0 4655 22487 4.830719656284 1109364.0 813851.0 56672.0 165562.0 11055.0 5718.0 0.0 278740.0 648289.0 73.4 5.1 14.9 1.0 0.5 0.0 25.1 58.4 43 43 43.00 38 47702652 26.9 21.3 21.8 29.9 0.0 35.0 23.5 smartseq 1057672 SRR2088238 SRP060416 SRS980256 SRX1082207 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810673: T75_P2_C3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810673 GSM1810673 T75_P2_C3_ILC3 116958065 2719955 2016-01-28 01:00:06 130491965 116958065 2719955 1 2719955 index:0,count:2719955,average:43,stdev:0 GSM1810673_r1 2.29 7.46 0.2 73002621 87543638 57392179 72494654 119.92 126.31 0 0 0 0 0 0 56.41 73.06 3074164 1005221 3074164 1005221 61.09 69.65 3074164 1088486 3074164 958270 11856705 16.24 6.67 0 14.93 0 1.00 0 0.38 0 0.00 0 33.10 0 1781896 0 43 0 41.71 0 1.34 0 0.00 0 1.20 0 0.00 0 279.77 0 0.32 0 181435 0 2719955 0 405977 0 27279 0 10410 0 0 0 900370 0 6 0 0 0 496 0 46853 0 980 0 48335 0 50.59 0 1375919 0 4663 54258 11.635856744585 2719955.0 1781896.0 181435.0 405977.0 27279.0 10410.0 0.0 900370.0 1375919.0 65.5 6.7 14.9 1.0 0.4 0.0 33.1 50.6 43 43 43.00 38 116958065 26.1 21.9 21.8 30.2 0.0 35.5 23.8 smartseq 1057690 SRR2088239 SRP060416 SRS980255 SRX1082208 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810674: T75_P2_C4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810674 GSM1810674 T75_P2_C4_ILC3 99990308 2325356 2016-01-28 01:00:06 111998056 99990308 2325356 1 2325356 index:0,count:2325356,average:43,stdev:0 GSM1810674_r1 2.77 6.27 0.25 68227466 85474077 53600573 70779003 125.28 132.05 0 0 0 0 0 0 60.04 77.45 2864266 992121 2864266 992121 65.93 73.48 2864266 1089448 2864266 941193 8140963 11.93 5.59 0 15.97 0 0.97 0 0.34 0 0.00 0 27.63 0 1652344 0 43 0 41.85 0 1.35 0 0.00 0 1.13 0 0.00 0 298.97 0 0.33 0 130037 0 2325356 0 371419 0 22659 0 7823 0 0 0 642530 0 2 0 0 0 470 0 48131 0 767 0 49370 0 55.09 0 1280925 0 6717 56699 8.441119547417 2325356.0 1652344.0 130037.0 371419.0 22659.0 7823.0 0.0 642530.0 1280925.0 71.1 5.6 16.0 1.0 0.3 0.0 27.6 55.1 43 43 43.00 38 99990308 26.1 22.2 22.3 29.4 0.0 35.6 24.0 smartseq 1057801 SRR2088240 SRP060416 SRS980254 SRX1082209 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810675: T75_P2_C5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810675 GSM1810675 T75_P2_C5_ILC3 109278394 2541358 2016-01-28 01:00:06 122873882 109278394 2541358 1 2541358 index:0,count:2541358,average:43,stdev:0 GSM1810675_r1 3.62 6.15 0.17 74491399 90945518 59727438 76140643 122.09 127.48 0 0 0 0 0 0 56.97 72.07 2978260 1029721 2978260 1029721 61.82 68.69 2978260 1117348 2978260 981301 12449964 16.71 5.63 0 14.90 0 0.97 0 0.50 0 0.00 0 27.41 0 1807337 0 43 0 41.81 0 1.31 0 0.00 0 1.16 0 0.00 0 295.13 0 0.34 0 143023 0 2541358 0 378652 0 24714 0 12699 0 0 0 696608 0 18 0 0 0 548 0 48477 0 898 0 49941 0 56.22 0 1428685 0 6632 56644 8.541013268999 2541358.0 1807337.0 143023.0 378652.0 24714.0 12699.0 0.0 696608.0 1428685.0 71.1 5.6 14.9 1.0 0.5 0.0 27.4 56.2 43 43 43.00 38 109278394 26.5 21.8 21.9 29.8 0.0 35.4 23.8 smartseq 1057817 SRR2088241 SRP060416 SRS980253 SRX1082210 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810676: T75_P2_C6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810676 GSM1810676 T75_P2_C6_ILC3 133899549 3113943 2016-01-28 01:00:06 149527117 133899549 3113943 1 3113943 index:0,count:3113943,average:43,stdev:0 GSM1810676_r1 2.23 5.71 0.31 97135482 120212756 76028290 98959440 123.76 130.16 0 0 0 0 0 0 58.25 75.3 4108234 1366638 4108234 1366638 64.07 71.33 4108234 1503324 4108234 1294699 13816252 14.22 4.75 0 17.06 0 0.98 0 0.41 0 0.00 0 23.25 0 2346331 0 43 0 41.89 0 1.49 0 0.00 0 1.19 0 0.00 0 339.70 0 0.32 0 148052 0 3113943 0 531309 0 30636 0 12922 0 0 0 724054 0 34 0 0 0 568 0 67798 0 991 0 69391 0 58.29 0 1815022 0 7218 80360 11.133277916320 3113943.0 2346331.0 148052.0 531309.0 30636.0 12922.0 0.0 724054.0 1815022.0 75.3 4.8 17.1 1.0 0.4 0.0 23.3 58.3 43 43 43.00 38 133899549 26.6 21.9 22.0 29.5 0.0 35.5 24.0 smartseq 1057833 SRR2088242 SRP060416 SRS980252 SRX1082211 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810677: T75_P2_C8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810677 GSM1810677 T75_P2_C8_ILC3 54747084 1273188 2016-01-28 01:00:06 61645692 54747084 1273188 1 1273188 index:0,count:1273188,average:43,stdev:0 GSM1810677_r1 4.15 6.44 0.21 40845371 50875493 33914509 43707045 124.56 128.87 0 0 0 0 0 0 59.57 72.71 1504839 589384 1504839 589384 63.14 69.16 1504839 624687 1504839 560598 6123598 14.99 4.31 0 14.04 0 1.11 0 0.65 0 0.00 0 20.53 0 989406 0 43 0 41.84 0 1.30 0 0.00 0 1.16 0 0.01 0 305.57 0 0.33 0 54891 0 1273188 0 178818 0 14187 0 8269 0 0 0 261326 0 2 0 0 0 199 0 21005 0 461 0 21667 0 63.67 0 810588 0 2652 24253 9.145173453997 1273188.0 989406.0 54891.0 178818.0 14187.0 8269.0 0.0 261326.0 810588.0 77.7 4.3 14.0 1.1 0.6 0.0 20.5 63.7 43 43 43.00 38 54747084 27.7 21.1 21.2 30.0 0.0 35.6 23.9 smartseq 1057849 SRR2088243 SRP060416 SRS980251 SRX1082212 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810678: T75_P2_C9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810678 GSM1810678 T75_P2_C9_ILC3 27257270 633890 2016-01-28 01:00:06 30795599 27257270 633890 1 633890 index:0,count:633890,average:43,stdev:0 GSM1810678_r1 5.69 6.81 0.23 18640691 23679494 14511388 19043216 127.03 131.23 0 0 0 0 0 0 56.85 74.13 734448 257660 734448 257660 63.99 70.49 734448 289984 734448 245035 2789468 14.96 5.51 0 16.66 0 0.95 0 0.49 0 0.00 0 27.06 0 453189 0 43 0 41.75 0 1.35 0 0.00 0 1.19 0 0.00 0 134.24 0 0.34 0 34925 0 633890 0 105593 0 6053 0 3090 0 0 0 171558 0 1 0 0 0 104 0 11088 0 195 0 11388 0 54.84 0 347596 0 3578 13053 3.648127445500 633890.0 453189.0 34925.0 105593.0 6053.0 3090.0 0.0 171558.0 347596.0 71.5 5.5 16.7 1.0 0.5 0.0 27.1 54.8 43 43 43.00 38 27257270 27.0 21.4 21.4 30.1 0.0 35.6 23.9 smartseq 1057866 SRR2088244 SRP060416 SRS980250 SRX1082213 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810679: T75_P2_D10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810679 GSM1810679 T75_P2_D10_ILC3 23100804 537228 2016-01-28 01:00:06 26112324 23100804 537228 1 537228 index:0,count:537228,average:43,stdev:0 GSM1810679_r1 2.91 7.12 0.32 14019104 16856685 10792443 13558986 120.24 125.63 0 0 0 0 0 0 55.25 73.17 598651 190090 598651 190090 61.76 70.53 598651 212500 598651 183229 2253106 16.07 6.89 0 15.69 0 1.14 0 0.32 0 0.00 0 34.49 0 344066 0 43 0 41.54 0 1.62 0 0.00 0 1.20 0 0.00 0 113.77 0 0.34 0 36995 0 537228 0 84274 0 6128 0 1744 0 0 0 185290 0 7 0 0 0 94 0 8046 0 179 0 8326 0 48.36 0 259792 0 2244 9480 4.224598930481 537228.0 344066.0 36995.0 84274.0 6128.0 1744.0 0.0 185290.0 259792.0 64.0 6.9 15.7 1.1 0.3 0.0 34.5 48.4 43 43 43.00 38 23100804 26.7 21.5 21.3 30.5 0.0 35.5 23.8 smartseq 1057882 SRR2088245 SRP060416 SRS980249 SRX1082214 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810680: T75_P2_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810680 GSM1810680 T75_P2_D11_NK 60806945 1414115 2016-01-28 01:00:06 68166804 60806945 1414115 1 1414115 index:0,count:1414115,average:43,stdev:0 GSM1810680_r1 2.19 6.01 0.21 42959357 55224609 33514136 44881267 128.55 133.92 0 0 0 0 0 0 62.99 81.75 1689761 656541 1689761 656541 70.22 77.09 1689761 731867 1689761 619113 5049155 11.75 5.06 0 16.91 0 1.01 0 0.32 0 0.00 0 24.96 0 1042243 0 43 0 41.73 0 1.45 0 0.00 0 1.17 0 0.00 0 363.63 0 0.32 0 71612 0 1414115 0 239134 0 14316 0 4543 0 0 0 353013 0 21 0 0 0 328 0 33180 0 436 0 33965 0 56.79 0 803109 0 4927 40939 8.309113050538 1414115.0 1042243.0 71612.0 239134.0 14316.0 4543.0 0.0 353013.0 803109.0 73.7 5.1 16.9 1.0 0.3 0.0 25.0 56.8 43 43 43.00 38 60806945 26.6 22.0 21.9 29.5 0.0 35.7 24.3 smartseq 1057897 SRR2088246 SRP060416 SRS980248 SRX1082215 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810681: T75_P2_D12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810681 GSM1810681 T75_P2_D12_ILC3 62906033 1462931 2016-01-28 01:00:06 70595760 62906033 1462931 1 1462931 index:0,count:1462931,average:43,stdev:0 GSM1810681_r1 2.2 8.77 0.29 34181180 39253529 26580694 32080819 114.84 120.69 0 0 0 0 0 0 50.68 66.79 1435065 428971 1435065 428971 54.71 64.16 1435065 463089 1435065 412076 6361560 18.61 8.09 0 13.96 0 1.25 0 0.37 0 0.00 0 40.51 0 846467 0 43 0 41.38 0 1.53 0 0.01 0 1.14 0 0.00 0 309.80 0 0.34 0 118293 0 1462931 0 204175 0 18283 0 5481 0 0 0 592700 0 22 0 0 0 247 0 16507 0 606 0 17382 0 43.90 0 642292 0 2208 18414 8.339673913043 1462931.0 846467.0 118293.0 204175.0 18283.0 5481.0 0.0 592700.0 642292.0 57.9 8.1 14.0 1.2 0.4 0.0 40.5 43.9 43 43 43.00 38 62906033 26.5 21.5 21.2 30.9 0.0 35.5 23.5 smartseq 1057913 SRR2088247 SRP060416 SRS980247 SRX1082216 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810682: T75_P2_D1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810682 GSM1810682 T75_P2_D1_ILC3 69317634 1612038 2016-01-28 01:00:06 77744037 69317634 1612038 1 1612038 index:0,count:1612038,average:43,stdev:0 GSM1810682_r1 2.75 6.16 0.19 47730393 59851320 38252357 50398547 125.39 131.75 0 0 0 0 0 0 59.63 75.35 1947799 688116 1947799 688116 64.24 71.25 1947799 741232 1947799 650652 6330293 13.26 5.52 0 14.93 0 0.92 0 0.42 0 0.00 0 27.08 0 1153901 0 43 0 41.89 0 1.32 0 0.00 0 1.17 0 0.00 0 276.35 0 0.32 0 89010 0 1612038 0 240655 0 14853 0 6728 0 0 0 436556 0 1 0 0 0 347 0 32219 0 561 0 33128 0 56.65 0 913246 0 5279 37524 7.108164425081 1612038.0 1153901.0 89010.0 240655.0 14853.0 6728.0 0.0 436556.0 913246.0 71.6 5.5 14.9 0.9 0.4 0.0 27.1 56.7 43 43 43.00 38 69317634 26.2 22.3 22.2 29.2 0.0 35.7 24.2 smartseq 1057928 SRR2088248 SRP060416 SRS980246 SRX1082217 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810683: T75_P2_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810683 GSM1810683 T75_P2_D2_NK 37472479 871453 2016-01-28 01:00:06 42214337 37472479 871453 1 871453 index:0,count:871453,average:43,stdev:0 GSM1810683_r1 2.61 7.4 0.22 22747817 27426029 17567549 22418857 120.57 127.62 0 0 0 0 0 0 57.92 76.38 985133 322295 985133 322295 62.7 72.6 985133 348884 985133 306361 3278102 14.41 7.02 0 15.44 0 1.01 0 0.38 0 0.00 0 34.75 0 556476 0 43 0 41.63 0 1.39 0 0.00 0 1.22 0 0.00 0 174.29 0 0.32 0 61149 0 871453 0 134513 0 8821 0 3330 0 0 0 302826 0 32 0 0 0 150 0 14766 0 280 0 15228 0 48.42 0 421963 0 3557 16951 4.765532752319 871453.0 556476.0 61149.0 134513.0 8821.0 3330.0 0.0 302826.0 421963.0 63.9 7.0 15.4 1.0 0.4 0.0 34.7 48.4 43 43 43.00 38 37472479 26.1 21.9 21.7 30.3 0.0 35.6 23.9 smartseq 1057944 SRR2088249 SRP060416 SRS980245 SRX1082218 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810684: T75_P2_D3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810684 GSM1810684 T75_P2_D3_ILC3 60794733 1413831 2016-01-28 01:00:06 68072202 60794733 1413831 1 1413831 index:0,count:1413831,average:43,stdev:0 GSM1810684_r1 2.57 6.18 0.19 40490970 50021071 32179143 41609296 123.54 129.31 0 0 0 0 0 0 60.84 77.63 1644446 597716 1644446 597716 66.17 73.79 1644446 650011 1644446 568186 5080302 12.55 5.86 0 15.02 0 1.02 0 0.40 0 0.00 0 29.10 0 982394 0 43 0 41.79 0 1.44 0 0.00 0 1.22 0 0.00 0 318.11 0 0.31 0 82821 0 1413831 0 212413 0 14454 0 5607 0 0 0 411376 0 7 0 0 0 244 0 28062 0 478 0 28791 0 54.46 0 769981 0 4983 31945 6.410796708810 1413831.0 982394.0 82821.0 212413.0 14454.0 5607.0 0.0 411376.0 769981.0 69.5 5.9 15.0 1.0 0.4 0.0 29.1 54.5 43 43 43.00 38 60794733 26.4 22.1 22.0 29.6 0.0 35.7 24.3 smartseq 1058088 SRR2088252 SRP060416 SRS979772 SRX1082221 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810687: T75_P2_D7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810687 GSM1810687 T75_P2_D7_ILC3 78945463 1835941 2016-01-28 01:00:06 88517238 78945463 1835941 1 1835941 index:0,count:1835941,average:43,stdev:0 GSM1810687_r1 3.93 7.4 0.22 49266915 60857462 38310457 49900410 123.53 130.25 0 0 0 0 0 0 57.21 74.95 2071694 690458 2071694 690458 62.14 70.97 2071694 749843 2071694 653790 7664530 15.56 6.63 0 15.55 0 0.99 0 0.39 0 0.00 0 32.90 0 1206781 0 43 0 41.58 0 1.30 0 0.00 0 1.16 0 0.00 0 194.39 0 0.32 0 121638 0 1835941 0 285500 0 18114 0 7091 0 0 0 603955 0 31 0 0 0 319 0 30383 0 592 0 31325 0 50.18 0 921281 0 3377 35042 10.376665679597 1835941.0 1206781.0 121638.0 285500.0 18114.0 7091.0 0.0 603955.0 921281.0 65.7 6.6 15.6 1.0 0.4 0.0 32.9 50.2 43 43 43.00 38 78945463 26.5 21.6 21.4 30.5 0.0 35.5 23.7 smartseq 1058104 SRR2088253 SRP060416 SRS980242 SRX1082222 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810688: T75_P2_D8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810688 GSM1810688 T75_P2_D8_ILC3 31906000 742000 2016-01-28 01:00:06 35978498 31906000 742000 1 742000 index:0,count:742000,average:43,stdev:0 GSM1810688_r1 2.78 5.46 0.15 24978095 31212421 20830303 26963374 124.96 129.44 0 0 0 0 0 0 62.9 76.14 922789 378510 922789 378510 66.94 72.51 922789 402852 922789 360456 3222647 12.90 3.65 0 14.11 0 1.03 0 0.48 0 0.00 0 17.38 0 601803 0 43 0 41.90 0 1.42 0 0.00 0 1.20 0 0.00 0 178.08 0 0.31 0 27118 0 742000 0 104696 0 7640 0 3585 0 0 0 128972 0 16 0 0 0 190 0 16867 0 237 0 17310 0 67.00 0 497107 0 4015 19316 4.810958904110 742000.0 601803.0 27118.0 104696.0 7640.0 3585.0 0.0 128972.0 497107.0 81.1 3.7 14.1 1.0 0.5 0.0 17.4 67.0 43 43 43.00 38 31906000 27.5 21.6 21.6 29.3 0.0 35.7 24.4 smartseq 1058121 SRR2088254 SRP060416 SRS980241 SRX1082223 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810689: T75_P2_E10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810689 GSM1810689 T75_P2_E10_ILC3 44395350 1032450 2016-01-28 01:00:06 50062638 44395350 1032450 1 1032450 index:0,count:1032450,average:43,stdev:0 GSM1810689_r1 3.27 8.46 0.42 26688803 32723678 20671710 26656914 122.61 128.95 0 0 0 0 0 0 55.49 73.16 1111655 364466 1111655 364466 60.41 69.48 1111655 396755 1111655 346125 4429111 16.60 6.94 0 15.36 0 1.15 0 0.35 0 0.00 0 34.89 0 656799 0 43 0 41.49 0 1.52 0 0.00 0 1.20 0 0.00 0 232.30 0 0.33 0 71660 0 1032450 0 158602 0 11848 0 3595 0 0 0 360208 0 0 0 0 0 157 0 14975 0 366 0 15498 0 48.25 0 498197 0 2524 17386 6.888272583201 1032450.0 656799.0 71660.0 158602.0 11848.0 3595.0 0.0 360208.0 498197.0 63.6 6.9 15.4 1.1 0.3 0.0 34.9 48.3 43 43 43.00 38 44395350 26.5 21.4 21.4 30.6 0.0 35.5 23.7 smartseq 1058136 SRR2088255 SRP060416 SRS980240 SRX1082224 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810690: T75_P2_E11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810690 GSM1810690 T75_P2_E11_ILC3 101255239 2354773 2016-01-28 01:00:06 113029477 101255239 2354773 1 2354773 index:0,count:2354773,average:43,stdev:0 GSM1810690_r1 1.86 8.26 0.49 60294184 71685005 46602314 58128584 118.89 124.73 0 0 0 0 0 0 54.79 72.43 2553483 814657 2553483 814657 60.0 69.61 2553483 892125 2553483 782905 10696066 17.74 7.17 0 15.38 0 1.10 0 0.32 0 0.00 0 35.43 0 1486907 0 43 0 41.44 0 1.30 0 0.00 0 1.10 0 0.00 0 249.33 0 0.33 0 168828 0 2354773 0 362218 0 26003 0 7564 0 0 0 834299 0 2 0 0 0 300 0 33381 0 891 0 34574 0 47.76 0 1124689 0 2907 38188 13.136566907465 2354773.0 1486907.0 168828.0 362218.0 26003.0 7564.0 0.0 834299.0 1124689.0 63.1 7.2 15.4 1.1 0.3 0.0 35.4 47.8 43 43 43.00 38 101255239 26.8 21.2 21.1 30.9 0.0 35.5 23.6 smartseq 1058152 SRR2088256 SRP060416 SRS980238 SRX1082225 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810691: T75_P2_E1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810691 GSM1810691 T75_P2_E1_ILC3 127855039 2973373 2016-01-28 01:00:06 142226374 127855039 2973373 1 2973373 index:0,count:2973373,average:43,stdev:0 GSM1810691_r1 4.86 6.62 0.27 81377374 99661317 64332084 82449836 122.47 128.16 0 0 0 0 0 0 57.86 74.43 3338287 1146220 3338287 1146220 62.74 70.39 3338287 1242930 3338287 1084008 12427711 15.27 6.45 0 14.83 0 0.92 0 0.34 0 0.00 0 32.12 0 1981122 0 43 0 41.77 0 1.35 0 0.00 0 1.09 0 0.00 0 227.75 0 0.31 0 191805 0 2973373 0 441076 0 27274 0 9970 0 0 0 955007 0 22 0 0 0 511 0 49136 0 926 0 50595 0 51.79 0 1540046 0 4708 55335 11.753398470688 2973373.0 1981122.0 191805.0 441076.0 27274.0 9970.0 0.0 955007.0 1540046.0 66.6 6.5 14.8 0.9 0.3 0.0 32.1 51.8 43 43 43.00 38 127855039 26.2 21.9 21.9 30.0 0.0 35.6 24.0 smartseq 1058168 SRR2088257 SRP060416 SRS980239 SRX1082226 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810692: T75_P2_E2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810692 GSM1810692 T75_P2_E2_ILC3 71476320 1662240 2016-01-28 01:00:06 81193599 71476320 1662240 1 1662240 index:0,count:1662240,average:43,stdev:0 GSM1810692_r1 3.11 6.55 0.23 48271455 59213993 39165227 50485861 122.67 128.9 0 0 0 0 0 0 54.63 68.38 1893571 639125 1893571 639125 58.17 64.67 1893571 680500 1893571 604437 8566394 17.75 5.59 0 14.15 0 1.17 0 0.57 0 0.00 0 27.88 0 1169887 0 43 0 41.91 0 1.53 0 0.00 0 1.15 0 0.00 0 193.03 0 0.34 0 92997 0 1662240 0 235271 0 19411 0 9544 0 0 0 463398 0 20 0 0 0 227 0 26687 0 500 0 27434 0 56.23 0 934616 0 4627 30919 6.682299546142 1662240.0 1169887.0 92997.0 235271.0 19411.0 9544.0 0.0 463398.0 934616.0 70.4 5.6 14.2 1.2 0.6 0.0 27.9 56.2 43 43 43.00 38 71476320 26.6 21.5 21.8 30.1 0.0 35.3 23.8 smartseq 1058184 SRR2088258 SRP060416 SRS980237 SRX1082227 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810693: T75_P2_E3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810693 GSM1810693 T75_P2_E3_ILC3 112737959 2621813 2016-01-28 01:00:06 125481391 112737959 2621813 1 2621813 index:0,count:2621813,average:43,stdev:0 GSM1810693_r1 3.53 6.49 0.19 73307525 89394293 57211205 73403169 121.94 128.3 0 0 0 0 0 0 59.13 76.93 3102543 1054132 3102543 1054132 64.45 72.89 3102543 1148912 3102543 998861 9628745 13.13 6.26 0 15.73 0 0.91 0 0.34 0 0.00 0 30.75 0 1782658 0 43 0 41.75 0 1.45 0 0.00 0 1.13 0 0.00 0 286.02 0 0.31 0 164256 0 2621813 0 412322 0 23841 0 9033 0 0 0 806281 0 9 0 0 0 510 0 51301 0 775 0 52595 0 52.27 0 1370336 0 6760 58841 8.704289940828 2621813.0 1782658.0 164256.0 412322.0 23841.0 9033.0 0.0 806281.0 1370336.0 68.0 6.3 15.7 0.9 0.3 0.0 30.8 52.3 43 43 43.00 38 112737959 26.3 22.0 21.8 29.9 0.0 35.7 24.1 smartseq 1058201 SRR2088259 SRP060416 SRS980236 SRX1082228 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810694: T75_P2_E4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810694 GSM1810694 T75_P2_E4_ILC3 66703535 1551245 2016-01-28 01:00:06 76116843 66703535 1551245 1 1551245 index:0,count:1551245,average:43,stdev:0 GSM1810694_r1 3.11 6.96 0.21 42633766 51828476 31962380 41314238 121.57 129.26 0 0 0 0 0 0 55.19 74.91 1953819 574081 1953819 574081 61.47 70.49 1953819 639348 1953819 540203 6108442 14.33 6.45 0 17.65 0 0.98 0 0.35 0 0.00 0 31.62 0 1040143 0 43 0 41.71 0 1.29 0 0.00 0 1.12 0 0.00 0 206.83 0 0.36 0 100037 0 1551245 0 273766 0 15171 0 5381 0 0 0 490550 0 4 0 0 0 320 0 26884 0 444 0 27652 0 49.40 0 766377 0 5247 31925 6.084429197637 1551245.0 1040143.0 100037.0 273766.0 15171.0 5381.0 0.0 490550.0 766377.0 67.1 6.4 17.6 1.0 0.3 0.0 31.6 49.4 43 43 43.00 38 66703535 26.3 21.6 21.8 30.3 0.0 35.1 23.5 smartseq 1058315 SRR2088260 SRP060416 SRS980235 SRX1082229 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810695: T75_P2_E5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810695 GSM1810695 T75_P2_E5_ILC3 109779559 2553013 2016-01-28 01:00:06 122558995 109779559 2553013 1 2553013 index:0,count:2553013,average:43,stdev:0 GSM1810695_r1 5.28 6.1 0.16 74859387 93841490 58086711 76556180 125.36 131.8 0 0 0 0 0 0 58.63 76.6 3119192 1063868 3119192 1063868 65.42 73.14 3119192 1187018 3119192 1015804 9964486 13.31 5.61 0 16.67 0 0.90 0 0.33 0 0.00 0 27.70 0 1814514 0 43 0 41.82 0 1.39 0 0.00 0 1.14 0 0.00 0 278.51 0 0.32 0 143146 0 2553013 0 425706 0 23002 0 8380 0 0 0 707117 0 18 0 0 0 517 0 50953 0 788 0 52276 0 54.40 0 1388808 0 7067 59698 8.447431724919 2553013.0 1814514.0 143146.0 425706.0 23002.0 8380.0 0.0 707117.0 1388808.0 71.1 5.6 16.7 0.9 0.3 0.0 27.7 54.4 43 43 43.00 38 109779559 26.3 22.0 21.9 29.8 0.0 35.6 24.1 smartseq 1058329 SRR2088261 SRP060416 SRS980234 SRX1082230 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810696: T75_P2_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810696 GSM1810696 T75_P2_E6_NK 116740442 2714894 2016-01-28 01:00:06 129976416 116740442 2714894 1 2714894 index:0,count:2714894,average:43,stdev:0 GSM1810696_r1 2.36 6.36 0.23 79593723 97822368 63919170 82637340 122.9 129.28 0 0 0 0 0 0 56.4 71.3 3256702 1090227 3256702 1090227 60.81 67.82 3256702 1175311 3256702 1037076 12666407 15.91 5.61 0 14.87 0 0.97 0 0.39 0 0.00 0 27.44 0 1932909 0 43 0 41.80 0 1.45 0 0.00 0 1.19 0 0.00 0 287.46 0 0.31 0 152349 0 2714894 0 403768 0 26324 0 10564 0 0 0 745097 0 63 0 0 0 450 0 47205 0 968 0 48686 0 56.32 0 1529141 0 5297 54253 10.242212573155 2714894.0 1932909.0 152349.0 403768.0 26324.0 10564.0 0.0 745097.0 1529141.0 71.2 5.6 14.9 1.0 0.4 0.0 27.4 56.3 43 43 43.00 38 116740442 26.4 21.8 21.7 30.1 0.0 35.6 24.0 smartseq 1058346 SRR2088262 SRP060416 SRS980233 SRX1082231 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810697: T75_P2_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810697 GSM1810697 T75_P2_E7_NK 109667501 2550407 2016-01-28 01:00:06 122572841 109667501 2550407 1 2550407 index:0,count:2550407,average:43,stdev:0 GSM1810697_r1 2.69 7.38 0.21 65633659 79648607 49527252 63650276 121.35 128.52 0 0 0 0 0 0 54.47 73.66 2895225 879194 2895225 879194 60.9 70.41 2895225 982909 2895225 840453 10377887 15.81 7.13 0 16.48 0 1.06 0 0.30 0 0.00 0 35.36 0 1613949 0 43 0 41.49 0 1.28 0 0.00 0 1.14 0 0.00 0 229.54 0 0.34 0 181916 0 2550407 0 420310 0 27088 0 7633 0 0 0 901737 0 2 0 0 0 510 0 41561 0 940 0 43013 0 46.80 0 1193639 0 3661 48878 13.350996995356 2550407.0 1613949.0 181916.0 420310.0 27088.0 7633.0 0.0 901737.0 1193639.0 63.3 7.1 16.5 1.1 0.3 0.0 35.4 46.8 43 43 43.00 38 109667501 26.4 21.6 21.5 30.5 0.0 35.5 23.6 smartseq 1058361 SRR2088263 SRP060416 SRS980232 SRX1082232 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810698: T75_P2_E8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810698 GSM1810698 T75_P2_E8_ILC3 53724243 1249401 2016-01-28 01:00:06 60430313 53724243 1249401 1 1249401 index:0,count:1249401,average:43,stdev:0 GSM1810698_r1 5.75 5.39 0.24 41441901 51983071 34489904 44521339 125.44 129.09 0 0 0 0 0 0 63.91 77.74 1505177 641151 1505177 641151 68.31 74.42 1505177 685343 1505177 613781 5315577 12.83 3.83 0 14.29 0 0.93 0 0.46 0 0.00 0 18.31 0 1003264 0 43 0 41.82 0 1.34 0 0.00 0 1.26 0 0.01 0 299.86 0 0.31 0 47842 0 1249401 0 178492 0 11634 0 5751 0 0 0 228752 0 11 0 0 0 384 0 24592 0 461 0 25448 0 66.01 0 824772 0 3222 28495 8.843885785227 1249401.0 1003264.0 47842.0 178492.0 11634.0 5751.0 0.0 228752.0 824772.0 80.3 3.8 14.3 0.9 0.5 0.0 18.3 66.0 43 43 43.00 38 53724243 27.6 21.3 21.3 29.9 0.0 35.6 24.2 smartseq 1058376 SRR2088264 SRP060416 SRS980230 SRX1082233 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810699: T75_P2_E9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810699 GSM1810699 T75_P2_E9_ILC3 23780806 553042 2016-01-28 01:00:06 26887695 23780806 553042 1 553042 index:0,count:553042,average:43,stdev:0 GSM1810699_r1 5.17 6.7 0.17 15434060 19326771 11942102 15701119 125.22 131.48 0 0 0 0 0 0 59.17 77.81 657297 223084 657297 223084 65.09 74.11 657297 245402 657297 212481 2179886 14.12 6.22 0 16.34 0 1.07 0 0.34 0 0.00 0 30.41 0 377047 0 43 0 41.65 0 1.23 0 0.00 0 1.10 0 0.00 0 124.43 0 0.33 0 34373 0 553042 0 90342 0 5936 0 1881 0 0 0 168178 0 4 0 0 0 111 0 9560 0 166 0 9841 0 51.84 0 286705 0 3023 10835 3.584187892822 553042.0 377047.0 34373.0 90342.0 5936.0 1881.0 0.0 168178.0 286705.0 68.2 6.2 16.3 1.1 0.3 0.0 30.4 51.8 43 43 43.00 38 23780806 26.8 21.5 21.4 30.2 0.0 35.5 23.8 smartseq 1058392 SRR2088265 SRP060416 SRS980231 SRX1082234 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810700: T75_P2_F10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810700 GSM1810700 T75_P2_F10_ILC3 40462914 940998 2016-01-28 01:00:06 45772714 40462914 940998 1 940998 index:0,count:940998,average:43,stdev:0 GSM1810700_r1 2.08 7.13 0.26 27154291 33994328 21954069 28712743 125.19 130.79 0 0 0 0 0 0 57.57 72.43 1042631 381682 1042631 381682 61.57 69.3 1042631 408167 1042631 365197 4176199 15.38 5.59 0 14.45 0 1.32 0 0.39 0 0.00 0 27.84 0 662958 0 43 0 41.66 0 1.77 0 0.01 0 1.15 0 0.00 0 225.84 0 0.33 0 52625 0 940998 0 135997 0 12401 0 3673 0 0 0 261966 0 0 0 0 0 139 0 16393 0 361 0 16893 0 56.00 0 526961 0 2408 18281 7.591777408638 940998.0 662958.0 52625.0 135997.0 12401.0 3673.0 0.0 261966.0 526961.0 70.5 5.6 14.5 1.3 0.4 0.0 27.8 56.0 43 43 43.00 38 40462914 27.0 21.3 21.3 30.3 0.0 35.5 23.8 smartseq 1058408 SRR2088266 SRP060416 SRS980229 SRX1082235 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810701: T75_P2_F11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810701 GSM1810701 T75_P2_F11_ILC3 99147293 2305751 2016-01-28 01:00:06 111004547 99147293 2305751 1 2305751 index:0,count:2305751,average:43,stdev:0 GSM1810701_r1 3.94 7.49 0.28 61170504 73858124 47731637 60070714 120.74 125.85 0 0 0 0 0 0 55.43 72.53 2488313 834343 2488313 834343 60.44 69.49 2488313 909853 2488313 799355 10173321 16.63 6.77 0 15.40 0 1.04 0 0.32 0 0.00 0 33.35 0 1505331 0 43 0 41.50 0 1.23 0 0.00 0 1.16 0 0.00 0 52.54 0 0.33 0 156188 0 2305751 0 355057 0 24041 0 7313 0 0 0 769066 0 10 0 0 0 375 0 34190 0 983 0 35558 0 49.89 0 1150274 0 3511 39974 11.385360296212 2305751.0 1505331.0 156188.0 355057.0 24041.0 7313.0 0.0 769066.0 1150274.0 65.3 6.8 15.4 1.0 0.3 0.0 33.4 49.9 43 43 43.00 38 99147293 26.7 21.3 21.1 30.9 0.0 35.5 23.6 smartseq 1058425 SRR2088267 SRP060416 SRS980228 SRX1082236 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810702: T75_P2_F1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810702 GSM1810702 T75_P2_F1_ILC3 127098110 2955770 2016-01-28 01:00:06 141700111 127098110 2955770 1 2955770 index:0,count:2955770,average:43,stdev:0 GSM1810702_r1 5.19 6.94 0.23 84140923 105262346 67783704 88500367 125.1 130.56 0 0 0 0 0 0 60.6 76.42 3312018 1238917 3312018 1238917 64.97 73.02 3312018 1328416 3312018 1183727 11862230 14.10 5.88 0 14.33 0 0.91 0 0.39 0 0.00 0 29.52 0 2044579 0 43 0 41.81 0 1.32 0 0.00 0 1.11 0 0.00 0 332.52 0 0.32 0 173689 0 2955770 0 423422 0 27028 0 11600 0 0 0 872563 0 13 0 0 0 521 0 51332 0 1059 0 52925 0 54.85 0 1621157 0 4904 56756 11.573409461664 2955770.0 2044579.0 173689.0 423422.0 27028.0 11600.0 0.0 872563.0 1621157.0 69.2 5.9 14.3 0.9 0.4 0.0 29.5 54.8 43 43 43.00 38 127098110 26.4 21.8 21.8 30.0 0.0 35.6 24.0 smartseq 1058441 SRR2088268 SRP060416 SRS980226 SRX1082237 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810703: T75_P2_F2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810703 GSM1810703 T75_P2_F2_ILC3 61841138 1438166 2016-01-28 01:00:06 70585973 61841138 1438166 1 1438166 index:0,count:1438166,average:43,stdev:0 GSM1810703_r1 2.7 7.17 0.21 40840345 50210284 32298714 41459482 122.94 128.36 0 0 0 0 0 0 59.35 76.23 1666909 589149 1666909 589149 64.87 72.77 1666909 643862 1666909 562444 5811900 14.23 5.99 0 15.28 0 0.95 0 0.38 0 0.00 0 29.65 0 992617 0 43 0 41.79 0 1.32 0 0.00 0 1.12 0 0.00 0 323.59 0 0.34 0 86130 0 1438166 0 219759 0 13671 0 5472 0 0 0 426406 0 8 0 0 0 218 0 29582 0 445 0 30253 0 53.74 0 772858 0 5360 34533 6.442723880597 1438166.0 992617.0 86130.0 219759.0 13671.0 5472.0 0.0 426406.0 772858.0 69.0 6.0 15.3 1.0 0.4 0.0 29.6 53.7 43 43 43.00 38 61841138 26.5 21.6 21.9 30.0 0.0 35.1 23.6 smartseq 1058456 SRR2088269 SRP060416 SRS980227 SRX1082238 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810704: T75_P2_F3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810704 GSM1810704 T75_P2_F3_ILC3 104079049 2420443 2016-01-28 01:00:06 116496903 104079049 2420443 1 2420443 index:0,count:2420443,average:43,stdev:0 GSM1810704_r1 3.49 6.46 0.29 69148962 85169256 54869961 70644151 123.17 128.75 0 0 0 0 0 0 58.79 75.27 2805481 989269 2805481 989269 63.83 71.75 2805481 1074142 2805481 943092 10845890 15.68 5.90 0 15.22 0 1.00 0 0.40 0 0.00 0 29.08 0 1682844 0 43 0 41.75 0 1.28 0 0.00 0 1.11 0 0.00 0 272.30 0 0.32 0 142839 0 2420443 0 368489 0 24194 0 9640 0 0 0 703765 0 18 0 0 0 396 0 41104 0 887 0 42405 0 54.30 0 1314355 0 5178 48413 9.349748937814 2420443.0 1682844.0 142839.0 368489.0 24194.0 9640.0 0.0 703765.0 1314355.0 69.5 5.9 15.2 1.0 0.4 0.0 29.1 54.3 43 43 43.00 38 104079049 26.5 21.8 21.6 30.1 0.0 35.5 23.9 smartseq 1058568 SRR2088270 SRP060416 SRS980225 SRX1082239 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810705: T75_P2_F4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810705 GSM1810705 T75_P2_F4_ILC3 93601669 2176783 2016-01-28 01:00:06 104819698 93601669 2176783 1 2176783 index:0,count:2176783,average:43,stdev:0 GSM1810705_r1 4.47 5.5 0.34 66497477 82917264 54411603 70386846 124.69 129.36 0 0 0 0 0 0 60.58 74.91 2495893 971681 2495893 971681 64.96 70.88 2495893 1042007 2495893 919467 9122783 13.72 5.11 0 14.09 0 0.95 0 0.58 0 0.00 0 24.78 0 1604000 0 43 0 41.95 0 1.39 0 0.00 0 1.10 0 0.00 0 270.22 0 0.35 0 111273 0 2176783 0 306805 0 20771 0 12628 0 0 0 539384 0 10 0 0 0 383 0 48565 0 718 0 49676 0 59.59 0 1297195 0 7658 54758 7.150430921912 2176783.0 1604000.0 111273.0 306805.0 20771.0 12628.0 0.0 539384.0 1297195.0 73.7 5.1 14.1 1.0 0.6 0.0 24.8 59.6 43 43 43.00 38 93601669 26.4 22.1 22.1 29.4 0.0 35.6 24.3 smartseq 1058648 SRR2088275 SRP060416 SRS980220 SRX1082244 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810710: T75_P2_G12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810710 GSM1810710 T75_P2_G12_ILC3 109449104 2545328 2016-01-28 01:00:06 121972420 109449104 2545328 1 2545328 index:0,count:2545328,average:43,stdev:0 GSM1810710_r1 1.61 9.21 0.29 64783179 76955413 51346984 63901330 118.79 124.45 0 0 0 0 0 0 53.61 69.14 2612638 855598 2612638 855598 57.25 65.87 2612638 913722 2612638 815139 11978821 18.49 7.15 0 14.09 0 1.10 0 0.43 0 0.00 0 35.76 0 1596105 0 43 0 41.49 0 1.51 0 0.00 0 1.24 0 0.01 0 241.14 0 0.33 0 182055 0 2545328 0 358637 0 27967 0 10941 0 0 0 910315 0 1 0 0 0 440 0 35055 0 853 0 36349 0 48.62 0 1237468 0 2983 40932 13.721756620851 2545328.0 1596105.0 182055.0 358637.0 27967.0 10941.0 0.0 910315.0 1237468.0 62.7 7.2 14.1 1.1 0.4 0.0 35.8 48.6 43 43 43.00 38 109449104 26.6 21.3 21.0 31.1 0.0 35.6 23.6 smartseq 1058824 SRR2088280 SRP060416 SRS980217 SRX1082249 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810715: T75_P2_G8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810715 GSM1810715 T75_P2_G8_ILC3 61014721 1418947 2016-01-28 01:00:06 68604466 61014721 1418947 1 1418947 index:0,count:1418947,average:43,stdev:0 GSM1810715_r1 2.06 6.24 0.38 43357781 52378655 34798622 43736938 120.81 125.69 0 0 0 0 0 0 58.39 73.84 1720547 615987 1720547 615987 63.13 70.42 1720547 665998 1720547 587415 6494104 14.98 4.95 0 15.56 0 1.06 0 0.51 0 0.00 0 24.08 0 1055013 0 43 0 41.72 0 1.48 0 0.00 0 1.15 0 0.00 0 364.87 0 0.33 0 70291 0 1418947 0 220837 0 15015 0 7242 0 0 0 341677 0 3 0 0 0 325 0 26151 0 566 0 27045 0 58.79 0 834176 0 3685 31065 8.430122116689 1418947.0 1055013.0 70291.0 220837.0 15015.0 7242.0 0.0 341677.0 834176.0 74.4 5.0 15.6 1.1 0.5 0.0 24.1 58.8 43 43 43.00 38 61014721 27.2 21.4 21.4 30.0 0.0 35.6 23.9 smartseq 1058841 SRR2088281 SRP060416 SRS980214 SRX1082250 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810716: T75_P2_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810716 GSM1810716 T75_P2_G9_NK 21701928 504696 2016-01-28 01:00:06 24530864 21701928 504696 1 504696 index:0,count:504696,average:43,stdev:0 GSM1810716_r1 2.07 6.82 0.32 14152974 17103010 11221257 14201032 120.84 126.55 0 0 0 0 0 0 56.55 72.62 578405 195692 578405 195692 61.49 69.58 578405 212805 578405 187507 2413442 17.05 6.14 0 15.17 0 1.08 0 0.38 0 0.00 0 29.97 0 346053 0 43 0 41.64 0 1.30 0 0.00 0 1.14 0 0.00 0 121.13 0 0.34 0 31013 0 504696 0 76579 0 5438 0 1943 0 0 0 151262 0 7 0 0 0 117 0 7595 0 199 0 7918 0 53.39 0 269474 0 2582 9063 3.510069713400 504696.0 346053.0 31013.0 76579.0 5438.0 1943.0 0.0 151262.0 269474.0 68.6 6.1 15.2 1.1 0.4 0.0 30.0 53.4 43 43 43.00 38 21701928 27.0 21.3 21.2 30.5 0.0 35.5 23.7 smartseq 1058856 SRR2088282 SRP060416 SRS980215 SRX1082251 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810717: T75_P2_H11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810717 GSM1810717 T75_P2_H11_ILC3 96666021 2248047 2016-01-28 01:00:06 108692276 96666021 2248047 1 2248047 index:0,count:2248047,average:43,stdev:0 GSM1810717_r1 4.12 8.5 0.25 58497704 71849876 43800805 56093294 122.83 128.06 0 0 0 0 0 0 54.8 74.73 2492901 790253 2492901 790253 61.87 70.83 2492901 892238 2492901 749032 9258554 15.83 6.96 0 17.11 0 1.08 0 0.31 0 0.00 0 34.46 0 1442129 0 43 0 41.42 0 1.29 0 0.00 0 1.18 0 0.00 0 252.91 0 0.34 0 156420 0 2248047 0 384664 0 24313 0 6949 0 0 0 774656 0 11 0 0 0 440 0 38994 0 855 0 40300 0 47.04 0 1057465 0 4551 45145 9.919797846627 2248047.0 1442129.0 156420.0 384664.0 24313.0 6949.0 0.0 774656.0 1057465.0 64.2 7.0 17.1 1.1 0.3 0.0 34.5 47.0 43 43 43.00 38 96666021 26.2 21.4 21.2 31.1 0.0 35.3 23.4 smartseq 1058888 SRR2088284 SRP060416 SRS980212 SRX1082253 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810719: T75_P2_H2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810719 GSM1810719 T75_P2_H2_ILC3 39109876 909532 2016-01-28 01:00:06 45662791 39109876 909532 1 909532 index:0,count:909532,average:43,stdev:0 GSM1810719_r1 3.26 7.96 0.18 25527079 30542016 19714777 24656771 119.65 125.07 0 0 0 0 0 0 56.19 74.13 1075449 350641 1075449 350641 61.96 70.68 1075449 386604 1075449 334312 3895087 15.26 6.07 0 16.60 0 0.92 0 0.34 0 0.00 0 30.13 0 623976 0 43 0 41.68 0 1.51 0 0.00 0 1.18 0 0.00 0 272.86 0 0.41 0 55234 0 909532 0 150953 0 8413 0 3114 0 0 0 274029 0 3 0 0 0 126 0 14119 0 260 0 14508 0 52.01 0 473023 0 3650 16661 4.564657534247 909532.0 623976.0 55234.0 150953.0 8413.0 3114.0 0.0 274029.0 473023.0 68.6 6.1 16.6 0.9 0.3 0.0 30.1 52.0 43 43 43.00 38 39109876 26.3 20.8 22.0 30.9 0.0 34.4 22.6 smartseq 1058968 SRR2088289 SRP060416 SRS980207 SRX1082258 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810724: T75_P3_A12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810724 GSM1810724 T75_P3_A12_ILC2 31502445 732615 2016-01-28 01:00:06 36981767 31502445 732615 1 732615 index:0,count:732615,average:43,stdev:0 GSM1810724_r1 5.95 8.73 0.25 22146432 27588265 17061505 22238516 124.57 130.34 0 0 0 0 0 0 55.27 73.29 908020 301863 908020 301863 61.35 69.77 908020 335073 908020 287381 3595869 16.24 5.73 0 18.33 0 1.21 0 0.30 0 0.00 0 23.94 0 546175 0 43 0 41.42 0 1.21 0 0.01 0 1.11 0 0.00 0 155.14 0 0.44 0 41971 0 732615 0 134278 0 8874 0 2179 0 0 0 175387 0 5 0 0 0 92 0 12680 0 284 0 13061 0 56.22 0 411897 0 4280 14421 3.369392523364 732615.0 546175.0 41971.0 134278.0 8874.0 2179.0 0.0 175387.0 411897.0 74.6 5.7 18.3 1.2 0.3 0.0 23.9 56.2 43 43 43.00 38 31502445 26.7 20.1 21.2 32.0 0.0 34.1 22.1 smartseq 1059080 SRR2088290 SRP060416 SRS980206 SRX1082259 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810725: T75_P3_A3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810725 GSM1810725 T75_P3_A3_ILC2 82210238 1911866 2016-01-28 01:00:06 92809630 82210238 1911866 1 1911866 index:0,count:1911866,average:43,stdev:0 GSM1810725_r1 4.05 10.06 0.32 47926697 57394351 36276400 45546273 119.75 125.55 0 0 0 0 0 0 50.58 68.64 2043343 602054 2043343 602054 56.85 66.37 2043343 676709 2043343 582190 9463346 19.75 8.44 0 16.38 0 1.23 0 0.36 0 0.00 0 36.15 0 1190358 0 43 0 41.36 0 1.23 0 0.01 0 1.13 0 0.00 0 208.57 0 0.34 0 161321 0 1911866 0 313217 0 23435 0 6944 0 0 0 691129 0 3 0 0 0 181 0 22620 0 847 0 23651 0 45.88 0 877141 0 3535 26308 7.442149929279 1911866.0 1190358.0 161321.0 313217.0 23435.0 6944.0 0.0 691129.0 877141.0 62.3 8.4 16.4 1.2 0.4 0.0 36.1 45.9 43 43 43.00 38 82210238 25.9 21.0 21.1 32.1 0.0 35.2 23.3 smartseq 1059112 SRR2088292 SRP060416 SRS980204 SRX1082261 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810727: T75_P3_A7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810727 GSM1810727 T75_P3_A7_ILC2 29965754 696878 2016-01-28 01:00:06 35318722 29965754 696878 1 696878 index:0,count:696878,average:43,stdev:0 GSM1810727_r1 4.93 8.99 0.25 20372920 24821188 15661422 19913655 121.83 127.15 0 0 0 0 0 0 53.02 70.49 835608 266470 835608 266470 59.6 67.77 835608 299536 835608 256213 3503326 17.20 6.26 0 17.87 0 1.26 0 0.38 0 0.00 0 26.24 0 502605 0 43 0 41.43 0 1.20 0 0.01 0 1.10 0 0.00 0 167.25 0 0.45 0 43590 0 696878 0 124566 0 8746 0 2667 0 0 0 182860 0 9 0 0 0 73 0 10574 0 269 0 10925 0 54.25 0 378039 0 3650 11957 3.275890410959 696878.0 502605.0 43590.0 124566.0 8746.0 2667.0 0.0 182860.0 378039.0 72.1 6.3 17.9 1.3 0.4 0.0 26.2 54.2 43 43 43.00 38 29965754 26.5 20.1 21.6 31.8 0.0 34.0 22.0 smartseq 1059160 SRR2088295 SRP060416 SRS980200 SRX1082264 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810730: T75_P3_B3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810730 GSM1810730 T75_P3_B3_ILC2 148536405 3454335 2016-01-28 01:00:06 165517925 148536405 3454335 1 3454335 index:0,count:3454335,average:43,stdev:0 GSM1810730_r1 4.16 8.21 0.31 96313771 123166855 76239764 101744947 127.88 133.45 0 0 0 0 0 0 59.46 76.53 3790496 1400744 3790496 1400744 65.22 73.37 3790496 1536311 3790496 1342992 14411943 14.96 6.92 0 15.21 0 1.14 0 0.41 0 0.00 0 30.25 0 2355762 0 43 0 41.65 0 1.31 0 0.01 0 1.15 0 0.00 0 253.79 0 0.33 0 239043 0 3454335 0 525390 0 39502 0 13993 0 0 0 1045078 0 39 0 0 0 521 0 58792 0 1261 0 60613 0 52.99 0 1830372 0 5525 66841 12.097918552036 3454335.0 2355762.0 239043.0 525390.0 39502.0 13993.0 0.0 1045078.0 1830372.0 68.2 6.9 15.2 1.1 0.4 0.0 30.3 53.0 43 43 43.00 38 148536405 26.5 21.4 21.5 30.6 0.0 35.5 24.1 smartseq 1060952 SRR2088305 SRP060416 SRS980192 SRX1082274 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810740: T75_P3_C3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810740 GSM1810740 T75_P3_C3_ILC2 152945926 3556882 2016-01-28 01:00:06 169929391 152945926 3556882 1 3556882 index:0,count:3556882,average:43,stdev:0 GSM1810740_r1 5.96 8.16 0.25 95752507 124242945 74403576 101100840 129.75 135.88 0 0 0 0 0 0 56.81 74.62 3902023 1335976 3902023 1335976 63.19 71.27 3902023 1486225 3902023 1275977 15062256 15.73 7.40 0 15.78 0 1.16 0 0.33 0 0.00 0 32.39 0 2351825 0 43 0 41.56 0 1.20 0 0.01 0 1.12 0 0.00 0 297.79 0 0.33 0 263316 0 3556882 0 561398 0 41371 0 11593 0 0 0 1152093 0 24 0 0 0 295 0 52497 0 1388 0 54204 0 50.34 0 1790427 0 4379 60410 13.795387074675 3556882.0 2351825.0 263316.0 561398.0 41371.0 11593.0 0.0 1152093.0 1790427.0 66.1 7.4 15.8 1.2 0.3 0.0 32.4 50.3 43 43 43.00 38 152945926 26.2 21.5 21.5 30.7 0.0 35.5 24.0 smartseq 1060984 SRR2088307 SRP060416 SRS980190 SRX1082276 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810742: T75_P3_C5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810742 GSM1810742 T75_P3_C5_ILC2 134968701 3138807 2016-01-28 01:00:06 151352606 134968701 3138807 1 3138807 index:0,count:3138807,average:43,stdev:0 GSM1810742_r1 4.82 6.88 0.32 94303374 119541433 76535350 100001749 126.76 130.66 0 0 0 0 0 0 57.02 71.28 3518433 1303613 3518433 1303613 62.74 68.01 3518433 1434434 3518433 1243736 15098626 16.01 5.79 0 14.58 0 1.08 0 0.47 0 0.00 0 25.60 0 2286419 0 43 0 41.85 0 1.32 0 0.01 0 1.15 0 0.00 0 275.60 0 0.34 0 181822 0 3138807 0 457672 0 34000 0 14794 0 0 0 803594 0 19 0 0 0 402 0 52278 0 1163 0 53862 0 58.26 0 1828747 0 5948 62120 10.443846671150 3138807.0 2286419.0 181822.0 457672.0 34000.0 14794.0 0.0 803594.0 1828747.0 72.8 5.8 14.6 1.1 0.5 0.0 25.6 58.3 43 43 43.00 38 134968701 26.5 21.8 22.0 29.7 0.0 35.4 24.2 smartseq 1061002 SRR2088308 SRP060416 SRS980189 SRX1082277 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810743: T75_P3_C6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810743 GSM1810743 T75_P3_C6_ILC2 143430972 3335604 2016-01-28 01:00:06 160302285 143430972 3335604 1 3335604 index:0,count:3335604,average:43,stdev:0 GSM1810743_r1 6.0 8.11 0.29 91103756 116231094 69317099 92265994 127.58 133.11 0 0 0 0 0 0 57.96 77.64 3766079 1292731 3766079 1292731 65.81 74.25 3766079 1467860 3766079 1236254 12903237 14.16 7.17 0 16.96 0 1.09 0 0.35 0 0.00 0 31.69 0 2230515 0 43 0 41.63 0 1.17 0 0.01 0 1.13 0 0.00 0 235.45 0 0.35 0 239291 0 3335604 0 565588 0 36340 0 11632 0 0 0 1057117 0 1 0 0 0 254 0 51594 0 1232 0 53081 0 49.91 0 1664927 0 5137 61415 11.955421452209 3335604.0 2230515.0 239291.0 565588.0 36340.0 11632.0 0.0 1057117.0 1664927.0 66.9 7.2 17.0 1.1 0.3 0.0 31.7 49.9 43 43 43.00 38 143430972 26.2 21.6 21.7 30.5 0.0 35.4 23.9 smartseq 1061017 SRR2088309 SRP060416 SRS980188 SRX1082278 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810744: T75_P3_C7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810744 GSM1810744 T75_P3_C7_ILC2 133243369 3098683 2016-01-28 01:00:06 148705599 133243369 3098683 1 3098683 index:0,count:3098683,average:43,stdev:0 GSM1810744_r1 7.05 8.16 0.23 82157198 103563558 64209734 84255158 126.06 131.22 0 0 0 0 0 0 55.82 72.95 3267817 1127412 3267817 1127412 62.04 70.34 3267817 1252991 3267817 1087057 14286461 17.39 7.55 0 15.30 0 1.12 0 0.40 0 0.00 0 33.30 0 2019643 0 43 0 41.55 0 1.17 0 0.01 0 1.20 0 0.01 0 293.56 0 0.34 0 233982 0 3098683 0 474174 0 34783 0 12407 0 0 0 1031850 0 0 0 0 0 265 0 38826 0 1262 0 40353 0 49.88 0 1545469 0 3633 45671 12.571153316818 3098683.0 2019643.0 233982.0 474174.0 34783.0 12407.0 0.0 1031850.0 1545469.0 65.2 7.6 15.3 1.1 0.4 0.0 33.3 49.9 43 43 43.00 38 133243369 26.4 21.4 21.3 30.9 0.0 35.4 23.9 smartseq 1061130 SRR2088310 SRP060416 SRS980187 SRX1082279 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810745: T75_P3_C8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810745 GSM1810745 T75_P3_C8_ILC2 66745546 1552222 2016-01-28 01:00:06 75122789 66745546 1552222 1 1552222 index:0,count:1552222,average:43,stdev:0 GSM1810745_r1 4.76 7.9 0.3 44229803 53882411 35816518 45032018 121.82 125.73 0 0 0 0 0 0 54.95 69.1 1675766 593561 1675766 593561 59.83 66.28 1675766 646223 1675766 569333 8351474 18.88 6.52 0 14.25 0 1.19 0 0.49 0 0.00 0 28.74 0 1080118 0 43 0 41.70 0 1.24 0 0.01 0 1.07 0 0.00 0 254.00 0 0.33 0 101271 0 1552222 0 221187 0 18407 0 7589 0 0 0 446108 0 8 0 0 0 133 0 21588 0 592 0 22321 0 55.34 0 858931 0 3735 25537 6.837215528782 1552222.0 1080118.0 101271.0 221187.0 18407.0 7589.0 0.0 446108.0 858931.0 69.6 6.5 14.2 1.2 0.5 0.0 28.7 55.3 43 43 43.00 38 66745546 26.7 21.5 21.4 30.5 0.0 35.5 24.1 smartseq 1061146 SRR2088311 SRP060416 SRS980186 SRX1082280 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810746: T75_P3_C9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810746 GSM1810746 T75_P3_C9_ILC2 21603286 502402 2016-01-28 01:00:06 24412180 21603286 502402 1 502402 index:0,count:502402,average:43,stdev:0 GSM1810746_r1 4.77 6.08 0.27 16551808 21306133 13848598 18238987 128.72 131.7 0 0 0 0 0 0 63.19 76.4 579305 252340 579305 252340 68.34 73.27 579305 272912 579305 242015 2151017 13.00 4.30 0 13.74 0 1.03 0 0.46 0 0.00 0 19.02 0 399326 0 43 0 41.93 0 1.24 0 0.01 0 1.23 0 0.01 0 129.19 0 0.31 0 21618 0 502402 0 69038 0 5184 0 2322 0 0 0 95570 0 2 0 0 0 78 0 9598 0 186 0 9864 0 65.74 0 330288 0 3302 10930 3.310115081769 502402.0 399326.0 21618.0 69038.0 5184.0 2322.0 0.0 95570.0 330288.0 79.5 4.3 13.7 1.0 0.5 0.0 19.0 65.7 43 43 43.00 38 21603286 27.3 21.5 21.6 29.7 0.0 35.7 24.7 smartseq 1061163 SRR2088312 SRP060416 SRS980184 SRX1082281 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810747: T75_P3_D10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810747 GSM1810747 T75_P3_D10_ILC2 20382344 474008 2016-01-28 01:00:06 23059669 20382344 474008 1 474008 index:0,count:474008,average:43,stdev:0 GSM1810747_r1 4.1 6.74 0.32 14535888 18338028 11724849 15182991 126.16 129.49 0 0 0 0 0 0 58.91 74.03 539938 207544 539938 207544 65.39 70.51 539938 230372 539938 197692 2211228 15.21 5.39 0 15.17 0 1.07 0 0.49 0 0.00 0 24.12 0 352289 0 43 0 41.82 0 1.21 0 0.01 0 1.15 0 0.01 0 131.26 0 0.32 0 25560 0 474008 0 71920 0 5070 0 2306 0 0 0 114343 0 5 0 0 0 53 0 8581 0 154 0 8793 0 59.15 0 280369 0 3743 10201 2.725353994122 474008.0 352289.0 25560.0 71920.0 5070.0 2306.0 0.0 114343.0 280369.0 74.3 5.4 15.2 1.1 0.5 0.0 24.1 59.1 43 43 43.00 38 20382344 26.8 21.7 21.6 29.9 0.0 35.7 24.6 smartseq 1061178 SRR2088313 SRP060416 SRS980183 SRX1082282 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810748: T75_P3_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810748 GSM1810748 T75_P3_D11_NK 71552086 1664002 2016-01-28 01:00:06 80240803 71552086 1664002 1 1664002 index:0,count:1664002,average:43,stdev:0 GSM1810748_r1 2.81 6.83 0.39 48897012 60790078 39706768 51078595 124.32 128.64 0 0 0 0 0 0 56.28 70.43 1837108 670370 1837108 670370 61.48 67.35 1837108 732322 1837108 641084 8015875 16.39 6.01 0 14.37 0 1.11 0 0.55 0 0.00 0 26.76 0 1191076 0 43 0 41.71 0 1.25 0 0.01 0 1.21 0 0.01 0 181.53 0 0.33 0 99956 0 1664002 0 239190 0 18534 0 9114 0 0 0 445278 0 16 0 0 0 254 0 28355 0 661 0 29286 0 57.20 0 951886 0 4313 34155 7.919081845583 1664002.0 1191076.0 99956.0 239190.0 18534.0 9114.0 0.0 445278.0 951886.0 71.6 6.0 14.4 1.1 0.5 0.0 26.8 57.2 43 43 43.00 38 71552086 26.6 21.8 21.7 29.9 0.0 35.7 24.4 smartseq 1061196 SRR2088314 SRP060416 SRS980182 SRX1082283 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810749: T75_P3_D12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810749 GSM1810749 T75_P3_D12_ILC2 70071424 1629568 2016-01-28 01:00:06 78679080 70071424 1629568 1 1629568 index:0,count:1629568,average:43,stdev:0 GSM1810749_r1 4.04 7.38 0.35 45966866 56730053 36965216 47332394 123.42 128.05 0 0 0 0 0 0 55.43 70.14 1777522 622500 1777522 622500 59.88 66.56 1777522 672378 1777522 590734 7883495 17.15 6.63 0 14.45 0 1.18 0 0.48 0 0.00 0 29.43 0 1122960 0 43 0 41.65 0 1.28 0 0.01 0 1.15 0 0.00 0 209.52 0 0.35 0 108089 0 1629568 0 235442 0 19245 0 7859 0 0 0 479504 0 16 0 0 0 195 0 23434 0 637 0 24282 0 54.46 0 887518 0 4148 26782 6.456605593057 1629568.0 1122960.0 108089.0 235442.0 19245.0 7859.0 0.0 479504.0 887518.0 68.9 6.6 14.4 1.2 0.5 0.0 29.4 54.5 43 43 43.00 38 70071424 26.6 21.6 21.5 30.2 0.0 35.6 24.3 smartseq 1061213 SRR2088315 SRP060416 SRS980185 SRX1082284 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810750: T75_P3_D3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810750 GSM1810750 T75_P3_D3_ILC2 63535725 1477575 2016-01-28 01:00:06 71209089 63535725 1477575 1 1477575 index:0,count:1477575,average:43,stdev:0 GSM1810750_r1 3.25 8.68 0.26 37512839 45738030 29678250 37930781 121.93 127.81 0 0 0 0 0 0 54.76 70.8 1498472 506207 1498472 506207 60.08 68.72 1498472 555361 1498472 491339 6601619 17.60 8.22 0 14.17 0 1.06 0 0.31 0 0.00 0 36.07 0 924331 0 43 0 41.51 0 1.25 0 0.01 0 1.09 0 0.00 0 221.64 0 0.34 0 121388 0 1477575 0 209380 0 15689 0 4526 0 0 0 533029 0 6 0 0 0 166 0 19036 0 580 0 19788 0 48.39 0 714951 0 3373 21741 6.445597391047 1477575.0 924331.0 121388.0 209380.0 15689.0 4526.0 0.0 533029.0 714951.0 62.6 8.2 14.2 1.1 0.3 0.0 36.1 48.4 43 43 43.00 38 63535725 26.4 21.4 21.3 30.9 0.0 35.6 24.1 smartseq 1061228 SRR2088316 SRP060416 SRS980181 SRX1082285 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810751: T75_P3_D5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810751 GSM1810751 T75_P3_D5_ILC2 75879606 1764642 2016-01-28 01:00:06 85152466 75879606 1764642 1 1764642 index:0,count:1764642,average:43,stdev:0 GSM1810751_r1 7.52 7.37 0.23 49096438 62589979 39249398 51714423 127.48 131.76 0 0 0 0 0 0 58.41 74.36 1881497 700255 1881497 700255 64.46 71.25 1881497 772756 1881497 670927 7324576 14.92 6.94 0 14.58 0 1.03 0 0.37 0 0.00 0 30.66 0 1198877 0 43 0 41.68 0 1.19 0 0.01 0 1.15 0 0.00 0 176.46 0 0.33 0 122544 0 1764642 0 257209 0 18245 0 6442 0 0 0 541078 0 10 0 0 0 241 0 28654 0 620 0 29525 0 53.36 0 941668 0 5290 32559 6.154820415879 1764642.0 1198877.0 122544.0 257209.0 18245.0 6442.0 0.0 541078.0 941668.0 67.9 6.9 14.6 1.0 0.4 0.0 30.7 53.4 43 43 43.00 38 75879606 26.4 21.7 21.7 30.2 0.0 35.6 24.3 smartseq 1061244 SRR2088317 SRP060416 SRS980180 SRX1082286 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810752: T75_P3_D7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810752 GSM1810752 T75_P3_D7_ILC2 57949767 1347669 2016-01-28 01:00:06 65261612 57949767 1347669 1 1347669 index:0,count:1347669,average:43,stdev:0 GSM1810752_r1 7.23 7.62 0.3 37085068 46130499 29841201 38484172 124.39 128.96 0 0 0 0 0 0 57.61 73.07 1426034 524053 1426034 524053 62.7 70.79 1426034 570366 1426034 507669 6609949 17.82 7.01 0 14.29 0 1.04 0 0.32 0 0.00 0 31.14 0 909697 0 43 0 41.61 0 1.31 0 0.01 0 1.11 0 0.00 0 242.58 0 0.34 0 94431 0 1347669 0 192523 0 14013 0 4353 0 0 0 419606 0 15 0 0 0 110 0 15059 0 514 0 15698 0 53.22 0 717174 0 2481 17426 7.023780733575 1347669.0 909697.0 94431.0 192523.0 14013.0 4353.0 0.0 419606.0 717174.0 67.5 7.0 14.3 1.0 0.3 0.0 31.1 53.2 43 43 43.00 38 57949767 26.6 21.3 21.2 30.9 0.0 35.5 24.0 smartseq 1061260 SRR2088318 SRP060416 SRS980179 SRX1082287 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810753: T75_P3_D8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810753 GSM1810753 T75_P3_D8_ILC2 32873414 764498 2016-01-28 01:00:06 37204659 32873414 764498 1 764498 index:0,count:764498,average:43,stdev:0 GSM1810753_r1 4.77 8.19 0.21 19865255 24775551 15904636 20710721 124.72 130.22 0 0 0 0 0 0 56.54 72.18 772117 276352 772117 276352 61.41 69.7 772117 300156 772117 266842 3378022 17.00 7.79 0 13.85 0 1.10 0 0.42 0 0.00 0 34.55 0 488772 0 43 0 41.54 0 1.30 0 0.01 0 1.15 0 0.01 0 125.10 0 0.34 0 59590 0 764498 0 105903 0 8413 0 3201 0 0 0 264112 0 3 0 0 0 80 0 10029 0 265 0 10377 0 50.08 0 382869 0 2840 11252 3.961971830986 764498.0 488772.0 59590.0 105903.0 8413.0 3201.0 0.0 264112.0 382869.0 63.9 7.8 13.9 1.1 0.4 0.0 34.5 50.1 43 43 43.00 38 32873414 26.5 21.6 21.4 30.6 0.0 35.4 23.9 smartseq 1061275 SRR2088319 SRP060416 SRS980178 SRX1082288 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810754: T75_P3_E10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810754 GSM1810754 T75_P3_E10_ILC2 50145052 1166164 2016-01-28 01:00:06 56690594 50145052 1166164 1 1166164 index:0,count:1166164,average:43,stdev:0 GSM1810754_r1 4.68 7.89 0.37 32450499 40426493 26801278 34783178 124.58 129.78 0 0 0 0 0 0 59.38 73.3 1210459 471392 1210459 471392 62.78 70.64 1210459 498323 1210459 454280 5182603 15.97 6.93 0 12.93 0 1.05 0 0.39 0 0.00 0 30.49 0 793807 0 43 0 41.68 0 1.28 0 0.01 0 1.13 0 0.01 0 209.91 0 0.32 0 80811 0 1166164 0 150729 0 12229 0 4524 0 0 0 355604 0 8 0 0 0 122 0 15521 0 424 0 16075 0 55.14 0 643078 0 2886 16760 5.807345807346 1166164.0 793807.0 80811.0 150729.0 12229.0 4524.0 0.0 355604.0 643078.0 68.1 6.9 12.9 1.0 0.4 0.0 30.5 55.1 43 43 43.00 38 50145052 26.6 21.5 21.4 30.5 0.0 35.5 24.0 smartseq 1061387 SRR2088320 SRP060416 SRS980177 SRX1082289 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810755: T75_P3_E12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810755 GSM1810755 T75_P3_E12_ILC2 127309541 2960687 2016-01-28 01:00:06 142320431 127309541 2960687 1 2960687 index:0,count:2960687,average:43,stdev:0 GSM1810755_r1 4.54 7.66 0.28 85634212 107260108 69571794 90278991 125.25 129.76 0 0 0 0 0 0 59.65 74.68 3199549 1245961 3199549 1245961 64.14 71.22 3199549 1339856 3199549 1188158 13316042 15.55 6.32 0 14.20 0 1.13 0 0.43 0 0.00 0 27.88 0 2088870 0 43 0 41.70 0 1.22 0 0.01 0 1.17 0 0.00 0 280.49 0 0.33 0 187086 0 2960687 0 420484 0 33526 0 12803 0 0 0 825488 0 3 0 0 0 352 0 51774 0 1195 0 53324 0 56.35 0 1668386 0 5341 59800 11.196405167572 2960687.0 2088870.0 187086.0 420484.0 33526.0 12803.0 0.0 825488.0 1668386.0 70.6 6.3 14.2 1.1 0.4 0.0 27.9 56.4 43 43 43.00 38 127309541 26.3 21.8 21.7 30.2 0.0 35.5 24.2 smartseq 1061403 SRR2088321 SRP060416 SRS980176 SRX1082290 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810756: T75_P3_E2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810756 GSM1810756 T75_P3_E2_ILC2 37348725 868575 2016-01-28 01:00:06 42799188 37348725 868575 1 868575 index:0,count:868575,average:43,stdev:0 GSM1810756_r1 6.93 8.12 0.26 24187494 31115715 18988074 25555065 128.64 134.58 0 0 0 0 0 0 57.56 74.81 967114 341587 967114 341587 63.7 71.84 967114 378007 967114 328013 3828298 15.83 6.83 0 15.76 0 1.11 0 0.35 0 0.00 0 30.22 0 593446 0 43 0 41.59 0 1.27 0 0.01 0 1.15 0 0.00 0 223.35 0 0.37 0 59358 0 868575 0 136853 0 9605 0 3014 0 0 0 262510 0 6 0 0 0 79 0 11960 0 297 0 12342 0 52.57 0 456593 0 3381 13520 3.998816918072 868575.0 593446.0 59358.0 136853.0 9605.0 3014.0 0.0 262510.0 456593.0 68.3 6.8 15.8 1.1 0.3 0.0 30.2 52.6 43 43 43.00 38 37348725 26.6 21.2 21.3 31.0 0.0 35.1 23.5 smartseq 1061419 SRR2088322 SRP060416 SRS980175 SRX1082291 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810757: T75_P3_E3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810757 GSM1810757 T75_P3_E3_ILC2 117319867 2728369 2016-01-28 01:00:06 131075623 117319867 2728369 1 2728369 index:0,count:2728369,average:43,stdev:0 GSM1810757_r1 2.91 8.83 0.31 68069628 81252392 54299961 67759533 119.37 124.79 0 0 0 0 0 0 54.18 69.61 2707786 911606 2707786 911606 58.03 67.39 2707786 976402 2707786 882554 12805500 18.81 8.35 0 13.67 0 1.17 0 0.32 0 0.00 0 36.84 0 1682648 0 43 0 41.46 0 1.44 0 0.01 0 1.27 0 0.01 0 280.63 0 0.32 0 227925 0 2728369 0 372991 0 31906 0 8699 0 0 0 1005116 0 17 0 0 0 255 0 32148 0 1074 0 33494 0 48.00 0 1309657 0 3571 35985 10.077009241109 2728369.0 1682648.0 227925.0 372991.0 31906.0 8699.0 0.0 1005116.0 1309657.0 61.7 8.4 13.7 1.2 0.3 0.0 36.8 48.0 43 43 43.00 38 117319867 26.3 21.3 21.2 31.2 0.0 35.5 23.8 smartseq 1061435 SRR2088323 SRP060416 SRS980174 SRX1082292 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810758: T75_P3_E4_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810758 GSM1810758 T75_P3_E4_ILC2 75771160 1762120 2016-01-28 01:00:06 86662708 75771160 1762120 1 1762120 index:0,count:1762120,average:43,stdev:0 GSM1810758_r1 4.02 7.1 0.33 53005842 65704510 42785694 54881004 123.96 128.27 0 0 0 0 0 0 57.4 72.26 2013060 738849 2013060 738849 62.93 69.15 2013060 810015 2013060 707045 8374670 15.80 5.75 0 15.02 0 1.10 0 0.52 0 0.00 0 25.33 0 1287266 0 43 0 41.84 0 1.20 0 0.01 0 1.15 0 0.00 0 243.99 0 0.35 0 101299 0 1762120 0 264732 0 19385 0 9082 0 0 0 446387 0 39 0 0 0 291 0 31307 0 603 0 32240 0 58.03 0 1022534 0 5893 36672 6.222976412693 1762120.0 1287266.0 101299.0 264732.0 19385.0 9082.0 0.0 446387.0 1022534.0 73.1 5.7 15.0 1.1 0.5 0.0 25.3 58.0 43 43 43.00 38 75771160 26.8 21.3 21.7 30.2 0.0 35.0 23.7 smartseq 1061452 SRR2088324 SRP060416 SRS980173 SRX1082293 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810759: T75_P3_E5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810759 GSM1810759 T75_P3_E5_ILC2 130018197 3023679 2016-01-28 01:00:06 145427702 130018197 3023679 1 3023679 index:0,count:3023679,average:43,stdev:0 GSM1810759_r1 3.41 7.33 0.22 87754982 108912791 71518976 92097935 124.11 128.77 0 0 0 0 0 0 57.76 72.11 3326690 1234625 3326690 1234625 62.58 69.41 3326690 1337628 3326690 1188443 13634745 15.54 6.29 0 14.06 0 1.17 0 0.43 0 0.00 0 27.71 0 2137365 0 43 0 41.77 0 1.26 0 0.01 0 1.10 0 0.01 0 265.49 0 0.33 0 190097 0 3023679 0 425124 0 35335 0 12980 0 0 0 837999 0 19 0 0 0 291 0 46459 0 1353 0 48122 0 56.63 0 1712241 0 4602 52763 11.465232507605 3023679.0 2137365.0 190097.0 425124.0 35335.0 12980.0 0.0 837999.0 1712241.0 70.7 6.3 14.1 1.2 0.4 0.0 27.7 56.6 43 43 43.00 38 130018197 26.5 21.6 21.6 30.3 0.0 35.5 24.2 smartseq 1061467 SRR2088325 SRP060416 SRS980172 SRX1082294 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810760: T75_P3_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810760 GSM1810760 T75_P3_E6_NK 141264288 3285216 2016-01-28 01:00:06 157694499 141264288 3285216 1 3285216 index:0,count:3285216,average:43,stdev:0 GSM1810760_r1 4.3 8.74 0.26 86114889 107427064 64556873 84539882 124.75 130.95 0 0 0 0 0 0 54.44 74.3 3684603 1157342 3684603 1157342 61.49 70.54 3684603 1307067 3684603 1098854 14089335 16.36 7.77 0 17.29 0 1.21 0 0.30 0 0.00 0 33.78 0 2125794 0 43 0 41.44 0 1.14 0 0.01 0 1.15 0 0.00 0 236.54 0 0.35 0 255158 0 3285216 0 568072 0 39810 0 9790 0 0 0 1109822 0 32 0 0 0 479 0 54004 0 1411 0 55926 0 47.42 0 1557722 0 5463 63854 11.688449569833 3285216.0 2125794.0 255158.0 568072.0 39810.0 9790.0 0.0 1109822.0 1557722.0 64.7 7.8 17.3 1.2 0.3 0.0 33.8 47.4 43 43 43.00 38 141264288 25.6 21.5 21.7 31.2 0.0 35.4 23.6 smartseq 1061483 SRR2088326 SRP060416 SRS980171 SRX1082295 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810761: T75_P3_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810761 GSM1810761 T75_P3_E7_NK 129265869 3006183 2016-01-28 01:00:06 144790384 129265869 3006183 1 3006183 index:0,count:3006183,average:43,stdev:0 GSM1810761_r1 3.58 7.83 0.23 85663466 106128345 68096883 87533809 123.89 128.54 0 0 0 0 0 0 58.07 74.36 3321311 1216059 3321311 1216059 63.57 70.33 3321311 1331040 3321311 1150163 13320365 15.55 6.48 0 15.26 0 1.18 0 0.49 0 0.00 0 28.67 0 2093976 0 43 0 41.64 0 1.23 0 0.01 0 1.15 0 0.00 0 240.49 0 0.34 0 194746 0 3006183 0 458637 0 35545 0 14813 0 0 0 861849 0 8 0 0 0 427 0 58584 0 1144 0 60163 0 54.40 0 1635339 0 6480 69699 10.756018518519 3006183.0 2093976.0 194746.0 458637.0 35545.0 14813.0 0.0 861849.0 1635339.0 69.7 6.5 15.3 1.2 0.5 0.0 28.7 54.4 43 43 43.00 38 129265869 26.3 21.8 21.7 30.3 0.0 35.4 24.0 smartseq 1061499 SRR2088327 SRP060416 SRS980170 SRX1082296 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810762: T75_P3_E8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810762 GSM1810762 T75_P3_E8_ILC2 71445747 1661529 2016-01-28 01:00:06 80572673 71445747 1661529 1 1661529 index:0,count:1661529,average:43,stdev:0 GSM1810762_r1 3.3 8.86 0.24 42316899 51066805 33447674 42190449 120.68 126.14 0 0 0 0 0 0 56.34 73.02 1703071 589127 1703071 589127 60.92 70.66 1703071 637023 1703071 570100 7472155 17.66 8.00 0 14.37 0 1.21 0 0.32 0 0.00 0 35.55 0 1045597 0 43 0 41.46 0 1.37 0 0.01 0 1.17 0 0.01 0 170.90 0 0.34 0 133001 0 1661529 0 238825 0 20024 0 5291 0 0 0 590617 0 0 0 0 0 140 0 18884 0 654 0 19678 0 48.56 0 806772 0 2464 21441 8.701704545455 1661529.0 1045597.0 133001.0 238825.0 20024.0 5291.0 0.0 590617.0 806772.0 62.9 8.0 14.4 1.2 0.3 0.0 35.5 48.6 43 43 43.00 38 71445747 26.6 21.2 21.0 31.2 0.0 35.4 23.6 smartseq 1061515 SRR2088328 SRP060416 SRS980168 SRX1082297 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810763: T75_P3_E9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810763 GSM1810763 T75_P3_E9_ILC2 23206584 539688 2016-01-28 01:00:06 26262610 23206584 539688 1 539688 index:0,count:539688,average:43,stdev:0 GSM1810763_r1 3.96 5.36 0.19 19098078 24948802 16142166 21582871 130.64 133.7 0 0 0 0 0 0 66.69 79.58 651327 305817 651327 305817 71.67 76.07 651327 328651 651327 292334 1991549 10.43 3.07 0 13.76 0 0.93 0 0.51 0 0.00 0 13.60 0 458544 0 43 0 42.00 0 1.39 0 0.01 0 1.15 0 0.01 0 176.63 0 0.32 0 16582 0 539688 0 74252 0 4998 0 2775 0 0 0 73371 0 5 0 0 0 66 0 12082 0 168 0 12321 0 71.21 0 384292 0 3491 13864 3.971354912632 539688.0 458544.0 16582.0 74252.0 4998.0 2775.0 0.0 73371.0 384292.0 85.0 3.1 13.8 0.9 0.5 0.0 13.6 71.2 43 43 43.00 38 23206584 27.4 21.6 21.6 29.3 0.0 35.7 24.9 smartseq 1061531 SRR2088329 SRP060416 SRS979775 SRX1082298 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810764: T75_P3_F10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810764 GSM1810764 T75_P3_F10_ILC2 50299035 1169745 2016-01-28 01:00:06 56855231 50299035 1169745 1 1169745 index:0,count:1169745,average:43,stdev:0 GSM1810764_r1 5.61 7.17 0.22 35238404 45541115 28353310 37887698 129.24 133.63 0 0 0 0 0 0 63.06 79.57 1337643 540424 1337643 540424 69.54 76.43 1337643 595950 1337643 519041 4487570 12.73 5.78 0 15.20 0 1.02 0 0.35 0 0.00 0 25.37 0 856960 0 43 0 41.75 0 1.15 0 0.01 0 1.16 0 0.00 0 221.64 0 0.32 0 67624 0 1169745 0 177813 0 11940 0 4129 0 0 0 296716 0 12 0 0 0 150 0 22150 0 437 0 22749 0 58.06 0 679147 0 4450 25458 5.720898876404 1169745.0 856960.0 67624.0 177813.0 11940.0 4129.0 0.0 296716.0 679147.0 73.3 5.8 15.2 1.0 0.4 0.0 25.4 58.1 43 43 43.00 38 50299035 26.6 21.6 21.7 30.0 0.0 35.5 24.2 smartseq 1061642 SRR2088330 SRP060416 SRS980169 SRX1082299 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810765: T75_P3_F11_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810765 GSM1810765 T75_P3_F11_ILC2 136382197 3171679 2016-01-28 01:00:06 151972124 136382197 3171679 1 3171679 index:0,count:3171679,average:43,stdev:0 GSM1810765_r1 4.64 6.46 0.24 94665008 118542750 77506684 100233925 125.22 129.32 0 0 0 0 0 0 58.65 72.76 3502261 1350104 3502261 1350104 64.18 70.41 3502261 1477410 3502261 1306642 14979160 15.82 5.83 0 14.08 0 1.14 0 0.51 0 0.00 0 25.77 0 2302081 0 43 0 41.77 0 1.22 0 0.01 0 1.11 0 0.00 0 58.26 0 0.33 0 184994 0 3171679 0 446422 0 36266 0 16102 0 0 0 817230 0 24 0 0 0 356 0 47879 0 1156 0 49415 0 58.51 0 1855659 0 4143 55092 13.297610427227 3171679.0 2302081.0 184994.0 446422.0 36266.0 16102.0 0.0 817230.0 1855659.0 72.6 5.8 14.1 1.1 0.5 0.0 25.8 58.5 43 43 43.00 38 136382197 26.8 21.4 21.4 30.3 0.0 35.6 24.3 smartseq 1061657 SRR2088331 SRP060416 SRS980167 SRX1082300 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810766: T75_P3_F12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810766 GSM1810766 T75_P3_F12_ILC2 145971885 3394695 2016-01-28 01:00:06 162552605 145971885 3394695 1 3394695 index:0,count:3394695,average:43,stdev:0 GSM1810766_r1 5.12 7.5 0.29 101359432 130700612 80837921 107915100 128.95 133.5 0 0 0 0 0 0 60.96 77.59 3905520 1502616 3905520 1502616 67.47 73.91 3905520 1663116 3905520 1431344 13085982 12.91 5.84 0 15.56 0 1.15 0 0.38 0 0.00 0 25.86 0 2464868 0 43 0 41.74 0 1.28 0 0.01 0 1.17 0 0.00 0 284.21 0 0.32 0 198114 0 3394695 0 528251 0 39016 0 12861 0 0 0 877950 0 8 0 0 0 422 0 61294 0 1208 0 62932 0 57.05 0 1936617 0 5616 72745 12.953169515670 3394695.0 2464868.0 198114.0 528251.0 39016.0 12861.0 0.0 877950.0 1936617.0 72.6 5.8 15.6 1.1 0.4 0.0 25.9 57.0 43 43 43.00 38 145971885 26.4 21.9 21.9 29.8 0.0 35.6 24.3 smartseq 1061672 SRR2088332 SRP060416 SRS980166 SRX1082301 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810767: T75_P3_F2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810767 GSM1810767 T75_P3_F2_ILC2 43583682 1013574 2016-01-28 01:00:06 49905671 43583682 1013574 1 1013574 index:0,count:1013574,average:43,stdev:0 GSM1810767_r1 5.16 7.93 0.26 29105612 36732381 23379429 30621778 126.2 130.98 0 0 0 0 0 0 58.66 74.37 1108688 417272 1108688 417272 64.41 71.57 1108688 458211 1108688 401562 4382255 15.06 6.47 0 14.83 0 1.01 0 0.35 0 0.00 0 28.45 0 711389 0 43 0 41.67 0 1.21 0 0.01 0 1.15 0 0.00 0 228.05 0 0.36 0 65533 0 1013574 0 150301 0 10225 0 3579 0 0 0 288381 0 0 0 0 0 113 0 15887 0 349 0 16349 0 55.36 0 561088 0 4293 18385 4.282552993245 1013574.0 711389.0 65533.0 150301.0 10225.0 3579.0 0.0 288381.0 561088.0 70.2 6.5 14.8 1.0 0.4 0.0 28.5 55.4 43 43 43.00 38 43583682 26.6 21.3 21.6 30.5 0.0 35.1 23.6 smartseq 1061689 SRR2088333 SRP060416 SRS980165 SRX1082302 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810768: T75_P3_F3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810768 GSM1810768 T75_P3_F3_ILC2 134005157 3116399 2016-01-28 01:00:06 149469900 134005157 3116399 1 3116399 index:0,count:3116399,average:43,stdev:0 GSM1810768_r1 8.18 6.38 0.32 92161485 120677619 72888474 98635170 130.94 135.32 0 0 0 0 0 0 58.71 75.33 3559083 1313282 3559083 1313282 66.53 72.32 3559083 1488325 3559083 1260858 13224059 14.35 6.02 0 15.84 0 1.10 0 0.64 0 0.00 0 26.48 0 2237041 0 43 0 41.81 0 1.14 0 0.01 0 1.17 0 0.00 0 287.67 0 0.33 0 187576 0 3116399 0 493645 0 34390 0 19820 0 0 0 825148 0 32 0 0 0 478 0 52082 0 1160 0 53752 0 55.94 0 1743396 0 5947 62155 10.451488145283 3116399.0 2237041.0 187576.0 493645.0 34390.0 19820.0 0.0 825148.0 1743396.0 71.8 6.0 15.8 1.1 0.6 0.0 26.5 55.9 43 43 43.00 38 134005157 26.5 21.8 21.8 30.0 0.0 35.6 24.4 smartseq 1061706 SRR2088334 SRP060416 SRS980164 SRX1082303 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810769: T75_P3_F4_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810769 GSM1810769 T75_P3_F4_ILC2 132884964 3090348 2016-01-28 01:00:06 148312102 132884964 3090348 1 3090348 index:0,count:3090348,average:43,stdev:0 GSM1810769_r1 7.64 6.33 0.2 95888685 128463921 76115427 105347390 133.97 138.4 0 0 0 0 0 0 63.03 80.39 3632333 1460724 3632333 1460724 71.1 76.84 3632333 1647722 3632333 1396264 11294718 11.78 5.37 0 16.20 0 0.96 0 0.32 0 0.00 0 23.73 0 2317567 0 43 0 41.89 0 1.18 0 0.01 0 1.14 0 0.00 0 236.71 0 0.33 0 165896 0 3090348 0 500492 0 29578 0 9758 0 0 0 733445 0 31 0 0 0 423 0 60991 0 969 0 62414 0 58.80 0 1817075 0 6895 71923 10.431182015954 3090348.0 2317567.0 165896.0 500492.0 29578.0 9758.0 0.0 733445.0 1817075.0 75.0 5.4 16.2 1.0 0.3 0.0 23.7 58.8 43 43 43.00 38 132884964 26.3 22.2 22.3 29.3 0.0 35.6 24.5 smartseq 1061722 SRR2088335 SRP060416 SRS980162 SRX1082304 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810770: T75_P3_F5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810770 GSM1810770 T75_P3_F5_ILC2 159083273 3699611 2016-01-28 01:00:06 177085420 159083273 3699611 1 3699611 index:0,count:3699611,average:43,stdev:0 GSM1810770_r1 5.8 6.68 0.26 108475553 139482099 85897726 114498657 128.58 133.3 0 0 0 0 0 0 60.58 77.66 4218574 1593726 4218574 1593726 67.45 74.04 4218574 1774379 4218574 1519404 14684473 13.54 6.17 0 15.64 0 1.00 0 0.42 0 0.00 0 27.48 0 2630817 0 43 0 41.86 0 1.15 0 0.01 0 1.12 0 0.00 0 256.13 0 0.33 0 228092 0 3699611 0 578595 0 36900 0 15376 0 0 0 1016518 0 32 0 0 0 587 0 66955 0 1283 0 68857 0 55.47 0 2052222 0 5954 79190 13.300302317770 3699611.0 2630817.0 228092.0 578595.0 36900.0 15376.0 0.0 1016518.0 2052222.0 71.1 6.2 15.6 1.0 0.4 0.0 27.5 55.5 43 43 43.00 38 159083273 26.2 22.0 22.2 29.6 0.0 35.5 24.3 smartseq 1061737 SRR2088336 SRP060416 SRS980161 SRX1082305 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810771: T75_P3_F7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810771 GSM1810771 T75_P3_F7_ILC2 133985764 3115948 2016-01-28 01:00:06 149735625 133985764 3115948 1 3115948 index:0,count:3115948,average:43,stdev:0 GSM1810771_r1 7.06 7.55 0.22 89513887 115721721 72283109 96862964 129.28 134.0 0 0 0 0 0 0 59.75 75.26 3380836 1303901 3380836 1303901 65.42 72.36 3380836 1427510 3380836 1253640 13538984 15.13 6.47 0 14.43 0 1.07 0 0.36 0 0.00 0 28.53 0 2182136 0 43 0 41.72 0 1.22 0 0.01 0 1.14 0 0.00 0 249.28 0 0.34 0 201671 0 3115948 0 449580 0 33462 0 11219 0 0 0 889131 0 13 0 0 0 308 0 49732 0 1177 0 51230 0 55.60 0 1732556 0 5505 59491 10.806721162579 3115948.0 2182136.0 201671.0 449580.0 33462.0 11219.0 0.0 889131.0 1732556.0 70.0 6.5 14.4 1.1 0.4 0.0 28.5 55.6 43 43 43.00 38 133985764 26.3 21.9 21.9 30.0 0.0 35.5 24.1 smartseq 1061754 SRR2088337 SRP060416 SRS980163 SRX1082306 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810772: T75_P3_F8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810772 GSM1810772 T75_P3_F8_ILC2 76024344 1768008 2016-01-28 01:00:06 85641447 76024344 1768008 1 1768008 index:0,count:1768008,average:43,stdev:0 GSM1810772_r1 4.51 8.55 0.32 46625373 56809948 37255080 47123285 121.84 126.49 0 0 0 0 0 0 56.02 71.66 1807731 642437 1807731 642437 61.22 69.45 1807731 702124 1807731 622624 8389936 17.99 7.61 0 14.16 0 1.12 0 0.35 0 0.00 0 33.66 0 1146896 0 43 0 41.56 0 1.24 0 0.01 0 1.10 0 0.00 0 187.20 0 0.33 0 134501 0 1768008 0 250402 0 19822 0 6118 0 0 0 595172 0 4 0 0 0 224 0 23596 0 710 0 24534 0 50.71 0 896494 0 3335 26746 8.019790104948 1768008.0 1146896.0 134501.0 250402.0 19822.0 6118.0 0.0 595172.0 896494.0 64.9 7.6 14.2 1.1 0.3 0.0 33.7 50.7 43 43 43.00 38 76024344 26.6 21.3 21.3 30.9 0.0 35.4 23.7 smartseq 1061770 SRR2088338 SRP060416 SRS980159 SRX1082307 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810773: T75_P3_F9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810773 GSM1810773 T75_P3_F9_ILC2 20013533 465431 2016-01-28 01:00:06 22646407 20013533 465431 1 465431 index:0,count:465431,average:43,stdev:0 GSM1810773_r1 4.54 4.97 0.32 16767969 21569961 14347446 18749155 128.64 130.68 0 0 0 0 0 0 64.63 76.12 563850 259785 563850 259785 69.46 72.87 563850 279214 563850 248671 2226788 13.28 2.79 0 13.04 0 0.97 0 0.56 0 0.00 0 12.11 0 401964 0 43 0 42.04 0 1.34 0 0.01 0 1.13 0 0.00 0 167.56 0 0.31 0 12975 0 465431 0 60698 0 4510 0 2613 0 0 0 56344 0 7 0 0 0 71 0 9868 0 163 0 10109 0 73.32 0 341266 0 3294 11012 3.343047965999 465431.0 401964.0 12975.0 60698.0 4510.0 2613.0 0.0 56344.0 341266.0 86.4 2.8 13.0 1.0 0.6 0.0 12.1 73.3 43 43 43.00 38 20013533 27.4 21.6 21.7 29.2 0.0 35.7 25.0 smartseq 1061785 SRR2088339 SRP060416 SRS980160 SRX1082308 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810774: T75_P3_G10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810774 GSM1810774 T75_P3_G10_ILC2 43695482 1016174 2016-01-28 01:00:06 49220851 43695482 1016174 1 1016174 index:0,count:1016174,average:43,stdev:0 GSM1810774_r1 5.49 6.29 0.21 31915033 41154185 26047549 34510308 128.95 132.49 0 0 0 0 0 0 60.95 75.67 1163743 471116 1163743 471116 67.42 72.5 1163743 521077 1163743 451387 4331325 13.57 5.06 0 14.79 0 1.02 0 0.46 0 0.00 0 22.46 0 772924 0 43 0 41.83 0 1.18 0 0.01 0 1.10 0 0.00 0 215.19 0 0.32 0 51461 0 1016174 0 150297 0 10326 0 4691 0 0 0 228233 0 7 0 0 0 128 0 19287 0 348 0 19770 0 61.27 0 622627 0 5037 23001 4.566408576534 1016174.0 772924.0 51461.0 150297.0 10326.0 4691.0 0.0 228233.0 622627.0 76.1 5.1 14.8 1.0 0.5 0.0 22.5 61.3 43 43 43.00 38 43695482 26.8 21.7 21.7 29.8 0.0 35.7 24.6 smartseq 1061897 SRR2088340 SRP060416 SRS980158 SRX1082309 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810775: T75_P3_G12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810775 GSM1810775 T75_P3_G12_ILC2 131755139 3064073 2016-01-28 01:00:06 146398190 131755139 3064073 1 3064073 index:0,count:3064073,average:43,stdev:0 GSM1810775_r1 4.38 7.39 0.27 88758647 112926124 71391037 93961647 127.23 131.62 0 0 0 0 0 0 60.85 76.93 3390055 1317992 3390055 1317992 66.33 73.54 3390055 1436656 3390055 1259883 12985858 14.63 6.36 0 14.78 0 0.99 0 0.31 0 0.00 0 28.00 0 2166074 0 43 0 41.67 0 1.31 0 0.01 0 1.18 0 0.01 0 256.53 0 0.32 0 194782 0 3064073 0 452791 0 30377 0 9635 0 0 0 857987 0 38 0 0 0 451 0 53186 0 1166 0 54841 0 55.92 0 1713283 0 5130 60958 11.882651072125 3064073.0 2166074.0 194782.0 452791.0 30377.0 9635.0 0.0 857987.0 1713283.0 70.7 6.4 14.8 1.0 0.3 0.0 28.0 55.9 43 43 43.00 38 131755139 26.5 21.7 21.6 30.3 0.0 35.6 24.3 smartseq 1061992 SRR2088346 SRP060416 SRS980152 SRX1082315 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810781: T75_P3_G8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810781 GSM1810781 T75_P3_G8_ILC2 69862358 1624706 2016-01-28 01:00:06 78400242 69862358 1624706 1 1624706 index:0,count:1624706,average:43,stdev:0 GSM1810781_r1 5.38 7.48 0.27 47156535 60620449 38499097 51385047 128.55 133.47 0 0 0 0 0 0 60.46 75.31 1739453 695412 1739453 695412 65.78 72.88 1739453 756584 1739453 673012 6943054 14.72 6.26 0 13.96 0 1.15 0 0.44 0 0.00 0 27.62 0 1150226 0 43 0 41.69 0 1.33 0 0.01 0 1.15 0 0.01 0 265.86 0 0.32 0 101660 0 1624706 0 226789 0 18623 0 7177 0 0 0 448680 0 1 0 0 0 218 0 25237 0 644 0 26100 0 56.84 0 923437 0 4146 29093 7.017124939701 1624706.0 1150226.0 101660.0 226789.0 18623.0 7177.0 0.0 448680.0 923437.0 70.8 6.3 14.0 1.1 0.4 0.0 27.6 56.8 43 43 43.00 38 69862358 26.6 21.6 21.5 30.3 0.0 35.6 24.2 smartseq 1062008 SRR2088347 SRP060416 SRS980151 SRX1082316 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810782: T75_P3_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810782 GSM1810782 T75_P3_G9_NK 23728776 551832 2016-01-28 01:00:06 26737381 23728776 551832 1 551832 index:0,count:551832,average:43,stdev:0 GSM1810782_r1 3.15 4.69 0.29 19779798 25472747 16852630 22182632 128.78 131.63 0 0 0 0 0 0 63.39 74.99 671174 300490 671174 300490 68.14 71.63 671174 322979 671174 287024 2479032 12.53 2.80 0 13.28 0 1.06 0 0.60 0 0.00 0 12.44 0 474007 0 43 0 42.06 0 1.31 0 0.01 0 1.18 0 0.00 0 132.44 0 0.31 0 15444 0 551832 0 73284 0 5857 0 3301 0 0 0 68667 0 0 0 0 0 60 0 11636 0 194 0 11890 0 72.62 0 400723 0 3724 13765 3.696294307197 551832.0 474007.0 15444.0 73284.0 5857.0 3301.0 0.0 68667.0 400723.0 85.9 2.8 13.3 1.1 0.6 0.0 12.4 72.6 43 43 43.00 38 23728776 27.1 21.9 22.0 28.9 0.0 35.8 25.2 smartseq 1062024 SRR2088348 SRP060416 SRS980150 SRX1082317 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810783: T75_P3_H10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810783 GSM1810783 T75_P3_H10_ILC2 20041268 466076 2016-01-28 01:00:06 23534831 20041268 466076 1 466076 index:0,count:466076,average:43,stdev:0 GSM1810783_r1 4.86 7.73 0.22 14434659 18175804 11521265 15070757 125.92 130.81 0 0 0 0 0 0 56.12 71.51 555427 197767 555427 197767 62.02 68.55 555427 218585 555427 189585 2188818 15.16 5.32 0 16.28 0 1.39 0 0.76 0 0.00 0 22.24 0 352429 0 43 0 41.66 0 1.27 0 0.01 0 1.10 0 0.00 0 93.22 0 0.43 0 24782 0 466076 0 75855 0 6463 0 3522 0 0 0 103662 0 1 0 0 0 52 0 7826 0 166 0 8045 0 59.34 0 276574 0 3209 8737 2.722655032720 466076.0 352429.0 24782.0 75855.0 6463.0 3522.0 0.0 103662.0 276574.0 75.6 5.3 16.3 1.4 0.8 0.0 22.2 59.3 43 43 43.00 38 20041268 26.8 20.7 21.8 30.7 0.0 34.3 22.6 smartseq 1062040 SRR2088349 SRP060416 SRS980149 SRX1082318 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810784: T75_P3_H11_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810784 GSM1810784 T75_P3_H11_ILC2 146994597 3418479 2016-01-28 01:00:06 164860471 146994597 3418479 1 3418479 index:0,count:3418479,average:43,stdev:0 GSM1810784_r1 3.49 6.82 0.23 104388995 130768657 87830227 113290662 125.27 128.99 0 0 0 0 0 0 57.24 69.02 3675707 1448429 3675707 1448429 61.02 66.31 3675707 1544050 3675707 1391405 18222395 17.46 5.53 0 12.64 0 1.07 0 0.58 0 0.00 0 24.32 0 2530600 0 43 0 41.85 0 1.24 0 0.01 0 1.14 0 0.00 0 307.66 0 0.33 0 188988 0 3418479 0 432129 0 36492 0 19968 0 0 0 831419 0 25 0 0 0 448 0 53954 0 1257 0 55684 0 61.39 0 2098471 0 5183 61582 11.881535790083 3418479.0 2530600.0 188988.0 432129.0 36492.0 19968.0 0.0 831419.0 2098471.0 74.0 5.5 12.6 1.1 0.6 0.0 24.3 61.4 43 43 43.00 38 146994597 26.7 21.7 21.7 29.9 0.0 35.4 24.2 smartseq 1062152 SRR2088350 SRP060416 SRS980148 SRX1082319 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810785: T75_P3_H2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810785 GSM1810785 T75_P3_H2_ILC2 19450749 452343 2016-01-28 01:00:06 22814731 19450749 452343 1 452343 index:0,count:452343,average:43,stdev:0 GSM1810785_r1 4.92 8.66 0.26 13396322 16605181 10584074 13644871 123.95 128.92 0 0 0 0 0 0 57.35 73.97 523348 188508 523348 188508 63.36 71.11 523348 208266 523348 181224 2102719 15.70 6.11 0 16.33 0 1.13 0 0.37 0 0.00 0 25.83 0 328723 0 43 0 41.53 0 1.29 0 0.01 0 1.11 0 0.01 0 85.71 0 0.43 0 27616 0 452343 0 73875 0 5123 0 1656 0 0 0 116841 0 1 0 0 0 73 0 7404 0 196 0 7674 0 56.34 0 254848 0 3394 8312 2.449027695934 452343.0 328723.0 27616.0 73875.0 5123.0 1656.0 0.0 116841.0 254848.0 72.7 6.1 16.3 1.1 0.4 0.0 25.8 56.3 43 43 43.00 38 19450749 26.6 20.6 21.7 31.0 0.0 34.3 22.5 smartseq 1062168 SRR2088351 SRP060416 SRS980146 SRX1082320 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810786: T75_P3_H3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810786 GSM1810786 T75_P3_H3_ILC2 136720048 3179536 2016-01-28 01:00:06 153642519 136720048 3179536 1 3179536 index:0,count:3179536,average:43,stdev:0 GSM1810786_r1 4.47 7.1 0.24 95084343 120542277 77184025 100519406 126.77 130.23 0 0 0 0 0 0 59.8 74.77 3538195 1379948 3538195 1379948 65.48 71.31 3538195 1511084 3538195 1316045 13696987 14.41 5.94 0 14.53 0 1.03 0 0.43 0 0.00 0 25.96 0 2307563 0 43 0 41.82 0 1.24 0 0.01 0 1.15 0 0.00 0 272.53 0 0.33 0 188993 0 3179536 0 461997 0 32900 0 13605 0 0 0 825468 0 29 0 0 0 485 0 61970 0 1153 0 63637 0 58.05 0 1845566 0 6997 71675 10.243675861083 3179536.0 2307563.0 188993.0 461997.0 32900.0 13605.0 0.0 825468.0 1845566.0 72.6 5.9 14.5 1.0 0.4 0.0 26.0 58.0 43 43 43.00 38 136720048 26.3 21.9 22.0 29.8 0.0 35.4 24.1 smartseq 1062185 SRR2088352 SRP060416 SRS980145 SRX1082321 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810787: T75_P3_H4_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810787 GSM1810787 T75_P3_H4_ILC2 111779360 2599520 2016-01-28 01:00:06 125668249 111779360 2599520 1 2599520 index:0,count:2599520,average:43,stdev:0 GSM1810787_r1 5.09 6.28 0.2 80700401 103269461 68283435 89835776 127.97 131.56 0 0 0 0 0 0 63.95 76.5 2808163 1242680 2808163 1242680 68.29 73.78 2808163 1327054 2808163 1198403 10613399 13.15 5.45 0 12.26 0 0.93 0 0.44 0 0.00 0 23.88 0 1943172 0 43 0 42.04 0 1.40 0 0.01 0 1.14 0 0.00 0 292.45 0 0.32 0 141610 0 2599520 0 318804 0 24150 0 11338 0 0 0 620860 0 28 0 0 0 291 0 48036 0 747 0 49102 0 62.49 0 1624368 0 5838 55141 9.445186707777 2599520.0 1943172.0 141610.0 318804.0 24150.0 11338.0 0.0 620860.0 1624368.0 74.8 5.4 12.3 0.9 0.4 0.0 23.9 62.5 43 43 43.00 38 111779360 26.6 21.9 22.1 29.3 0.0 35.5 24.5 smartseq 1062201 SRR2088353 SRP060416 SRS980147 SRX1082322 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810788: T75_P3_H5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810788 GSM1810788 T75_P3_H5_ILC2 188146758 4375506 2016-01-28 01:00:06 209204003 188146758 4375506 1 4375506 index:0,count:4375506,average:43,stdev:0 GSM1810788_r1 5.46 5.46 0.3 150486063 195470672 125665379 166749497 129.89 132.69 0 0 0 0 0 0 61.27 73.96 5251552 2203861 5251552 2203861 67.31 70.38 5251552 2421185 5251552 2097128 20232535 13.44 3.65 0 14.10 0 0.93 0 0.60 0 0.00 0 16.27 0 3596802 0 43 0 42.17 0 1.21 0 0.01 0 1.16 0 0.00 0 437.55 0 0.32 0 159913 0 4375506 0 616884 0 40724 0 26186 0 0 0 711794 0 36 0 0 0 626 0 98212 0 1204 0 100078 0 68.10 0 2979918 0 10706 117072 10.935176536522 4375506.0 3596802.0 159913.0 616884.0 40724.0 26186.0 0.0 711794.0 2979918.0 82.2 3.7 14.1 0.9 0.6 0.0 16.3 68.1 43 43 43.00 38 188146758 26.5 22.4 22.6 28.6 0.0 35.6 24.9 smartseq 1062217 SRR2088354 SRP060416 SRS980144 SRX1082323 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810789: T75_P3_H8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810789 GSM1810789 T75_P3_H8_ILC2 104340016 2426512 2016-01-28 01:00:06 117859502 104340016 2426512 1 2426512 index:0,count:2426512,average:43,stdev:0 GSM1810789_r1 5.07 6.05 0.33 78184773 100714070 63464873 83892362 128.82 132.19 0 0 0 0 0 0 62.08 77.35 2862522 1169843 2862522 1169843 69.08 73.84 2862522 1301725 2862522 1116816 9983193 12.77 4.73 0 15.32 0 0.96 0 0.53 0 0.00 0 20.86 0 1884277 0 43 0 41.96 0 1.22 0 0.01 0 1.16 0 0.00 0 291.18 0 0.34 0 114807 0 2426512 0 371828 0 23235 0 12796 0 0 0 506204 0 24 0 0 0 414 0 52125 0 856 0 53419 0 62.33 0 1512449 0 7030 60759 8.642816500711 2426512.0 1884277.0 114807.0 371828.0 23235.0 12796.0 0.0 506204.0 1512449.0 77.7 4.7 15.3 1.0 0.5 0.0 20.9 62.3 43 43 43.00 38 104340016 26.8 21.9 22.1 29.3 0.0 35.4 24.4 smartseq 1062234 SRR2088355 SRP060416 SRS980143 SRX1082324 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810790: T75_P3_H9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810790 GSM1810790 T75_P3_H9_ILC2 34291296 797472 2016-01-28 01:00:06 38825973 34291296 797472 1 797472 index:0,count:797472,average:43,stdev:0 GSM1810790_r1 5.14 5.49 0.2 27777688 36120283 23482832 31135123 130.03 132.59 0 0 0 0 0 0 65.33 77.95 948467 435317 948467 435317 70.52 74.61 948467 469869 948467 416668 3177371 11.44 3.30 0 13.52 0 1.06 0 0.64 0 0.00 0 14.74 0 666310 0 43 0 42.05 0 1.17 0 0.01 0 1.16 0 0.01 0 220.84 0 0.32 0 26348 0 797472 0 107849 0 8488 0 5105 0 0 0 117569 0 14 0 0 0 117 0 16949 0 275 0 17355 0 70.03 0 558461 0 4387 19433 4.429678595851 797472.0 666310.0 26348.0 107849.0 8488.0 5105.0 0.0 117569.0 558461.0 83.6 3.3 13.5 1.1 0.6 0.0 14.7 70.0 43 43 43.00 38 34291296 27.3 21.7 21.9 29.1 0.0 35.6 24.9 smartseq 1062250 SRR2088356 SRP060416 SRS980142 SRX1082325 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810791: T75_P4_A10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810791 GSM1810791 T75_P4_A10_ILC1 19713264 458448 2016-01-28 01:00:06 22787028 19713264 458448 1 458448 index:0,count:458448,average:43,stdev:0 GSM1810791_r1 1.68 6.85 0.24 14193119 16881590 10736477 13649462 118.94 127.13 0 0 0 0 0 0 55.12 74.12 656893 191211 656893 191211 61.03 70.64 656893 211713 656893 182238 2124128 14.97 4.74 0 19.40 0 1.23 0 0.47 0 0.00 0 22.62 0 346914 0 43 0 41.62 0 1.44 0 0.00 0 1.13 0 0.00 0 165.04 0 0.35 0 21737 0 458448 0 88935 0 5656 0 2163 0 0 0 103715 0 3 0 0 0 94 0 7827 0 154 0 8078 0 56.27 0 257979 0 3183 9210 2.893496701225 458448.0 346914.0 21737.0 88935.0 5656.0 2163.0 0.0 103715.0 257979.0 75.7 4.7 19.4 1.2 0.5 0.0 22.6 56.3 43 43 43.00 38 19713264 27.1 20.6 21.5 30.7 0.0 34.9 23.3 smartseq 1062266 SRR2088357 SRP060416 SRS980141 SRX1082326 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810792: T75_P4_A11_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810792 GSM1810792 T75_P4_A11_ILC1 83503764 1941948 2016-01-28 01:00:06 93661028 83503764 1941948 1 1941948 index:0,count:1941948,average:43,stdev:0 GSM1810792_r1 1.94 6.0 0.28 61688162 77101980 47870674 63054645 124.99 131.72 0 0 0 0 0 0 58.73 76.64 2661944 877622 2661944 877622 64.9 72.53 2661944 969881 2661944 830578 7790348 12.63 4.46 0 17.99 0 1.07 0 0.45 0 0.00 0 21.53 0 1494386 0 43 0 41.81 0 1.34 0 0.00 0 1.14 0 0.00 0 268.89 0 0.31 0 86696 0 1941948 0 349300 0 20767 0 8735 0 0 0 418060 0 41 0 0 0 370 0 42666 0 675 0 43752 0 58.97 0 1145086 0 6805 50224 7.380455547392 1941948.0 1494386.0 86696.0 349300.0 20767.0 8735.0 0.0 418060.0 1145086.0 77.0 4.5 18.0 1.1 0.4 0.0 21.5 59.0 43 43 43.00 38 83503764 26.5 21.8 22.1 29.6 0.0 35.7 24.5 smartseq 1062282 SRR2088358 SRP060416 SRS980140 SRX1082327 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810793: T75_P4_A1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810793 GSM1810793 T75_P4_A1_ILC1 50424595 1172665 2016-01-28 01:00:06 57880241 50424595 1172665 1 1172665 index:0,count:1172665,average:43,stdev:0 GSM1810793_r1 1.32 7.96 0.2 32587040 37532083 24355253 30139538 115.17 123.75 0 0 0 0 0 0 50.17 68.58 1546200 402651 1546200 402651 54.91 64.73 1546200 440671 1546200 380023 5830034 17.89 6.29 0 18.38 0 1.21 0 0.43 0 0.00 0 29.92 0 802600 0 43 0 41.48 0 1.45 0 0.00 0 1.12 0 0.00 0 60.31 0 0.35 0 73772 0 1172665 0 215513 0 14196 0 5043 0 0 0 350826 0 4 0 0 0 209 0 15908 0 443 0 16564 0 50.06 0 587087 0 4141 18896 4.563148997827 1172665.0 802600.0 73772.0 215513.0 14196.0 5043.0 0.0 350826.0 587087.0 68.4 6.3 18.4 1.2 0.4 0.0 29.9 50.1 43 43 43.00 38 50424595 26.5 20.8 21.5 31.2 0.0 34.9 23.1 smartseq 1062298 SRR2088359 SRP060416 SRS980139 SRX1082328 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810794: T75_P4_A5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810794 GSM1810794 T75_P4_A5_ILC1 77526377 1802939 2016-01-28 01:00:06 86832145 77526377 1802939 1 1802939 index:0,count:1802939,average:43,stdev:0 GSM1810794_r1 1.83 6.29 0.18 51579579 59590701 39190453 48399137 115.53 123.5 0 0 0 0 0 0 50.17 67.24 2442265 631571 2442265 631571 56.44 64.81 2442265 710481 2442265 608792 9523339 18.46 5.82 0 17.73 0 1.24 0 0.46 0 0.00 0 28.47 0 1258916 0 43 0 41.72 0 1.37 0 0.00 0 1.18 0 0.00 0 295.03 0 0.31 0 105008 0 1802939 0 319635 0 22333 0 8324 0 0 0 513366 0 7 0 0 0 350 0 23980 0 641 0 24978 0 52.10 0 939281 0 4379 28633 6.538707467458 1802939.0 1258916.0 105008.0 319635.0 22333.0 8324.0 0.0 513366.0 939281.0 69.8 5.8 17.7 1.2 0.5 0.0 28.5 52.1 43 43 43.00 38 77526377 26.7 21.2 21.5 30.6 0.0 35.6 24.2 smartseq 1062410 SRR2088360 SRP060416 SRS980138 SRX1082329 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810795: T75_P4_A8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810795 GSM1810795 T75_P4_A8_ILC1 38313473 891011 2016-01-28 01:00:06 43137947 38313473 891011 1 891011 index:0,count:891011,average:43,stdev:0 GSM1810795_r1 2.28 4.17 0.19 32639707 41242572 26414161 35339180 126.36 133.79 0 0 0 0 0 0 64.24 79.95 1375519 502605 1375519 502605 70.09 76.68 1375519 548367 1375519 482033 3372648 10.33 2.35 0 17.26 0 1.07 0 0.48 0 0.00 0 10.64 0 782397 0 43 0 42.02 0 1.44 0 0.00 0 1.15 0 0.00 0 229.12 0 0.29 0 20944 0 891011 0 153756 0 9573 0 4264 0 0 0 94777 0 4 0 0 0 190 0 20940 0 278 0 21412 0 70.55 0 628641 0 5216 24364 4.671012269939 891011.0 782397.0 20944.0 153756.0 9573.0 4264.0 0.0 94777.0 628641.0 87.8 2.4 17.3 1.1 0.5 0.0 10.6 70.6 43 43 43.00 38 38313473 27.4 21.7 22.1 28.8 0.0 35.8 25.1 smartseq 1062426 SRR2088361 SRP060416 SRS980137 SRX1082330 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810796: T75_P4_A9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810796 GSM1810796 T75_P4_A9_ILC1 21523693 500551 2016-01-28 01:00:06 23913756 21523693 500551 1 500551 index:0,count:500551,average:43,stdev:0 GSM1810796_r1 2.04 4.5 0.21 16174384 19727672 12250395 16065878 121.97 131.15 0 0 0 0 0 0 57.18 76.33 776907 223334 776907 223334 63.81 72.51 776907 249235 776907 212140 2028209 12.54 4.27 0 19.58 0 1.02 0 0.42 0 0.00 0 20.53 0 390579 0 43 0 41.87 0 1.48 0 0.00 0 1.14 0 0.01 0 150.17 0 0.29 0 21363 0 500551 0 98003 0 5106 0 2083 0 0 0 102783 0 4 0 0 0 93 0 10056 0 182 0 10335 0 58.45 0 292576 0 4062 11681 2.875677006401 500551.0 390579.0 21363.0 98003.0 5106.0 2083.0 0.0 102783.0 292576.0 78.0 4.3 19.6 1.0 0.4 0.0 20.5 58.5 43 43 43.00 38 21523693 26.5 22.1 22.0 29.4 0.0 36.2 25.3 smartseq 1062442 SRR2088362 SRP060416 SRS980135 SRX1082331 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810797: T75_P4_B10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810797 GSM1810797 T75_P4_B10_ILC1 24870082 578374 2016-01-28 01:00:06 28924301 24870082 578374 1 578374 index:0,count:578374,average:43,stdev:0 GSM1810797_r1 2.08 6.73 0.25 17633301 20808609 13144783 16672721 118.01 126.84 0 0 0 0 0 0 53.08 72.44 845471 229126 845471 229126 59.29 69.0 845471 255934 845471 218248 2643088 14.99 5.03 0 19.95 0 1.28 0 0.41 0 0.00 0 23.68 0 431681 0 43 0 41.56 0 1.41 0 0.00 0 1.13 0 0.00 0 115.67 0 0.38 0 29084 0 578374 0 115392 0 7393 0 2361 0 0 0 136939 0 1 0 0 0 116 0 9591 0 229 0 9937 0 54.69 0 316289 0 3756 11117 2.959797657082 578374.0 431681.0 29084.0 115392.0 7393.0 2361.0 0.0 136939.0 316289.0 74.6 5.0 20.0 1.3 0.4 0.0 23.7 54.7 43 43 43.00 38 24870082 26.6 20.7 22.0 30.7 0.0 34.7 23.0 smartseq 1062459 SRR2088363 SRP060416 SRS980136 SRX1082332 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810798: T75_P4_B12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810798 GSM1810798 T75_P4_B12_ILC1 142521866 3314462 2016-01-28 01:00:06 156570219 142521866 3314462 1 3314462 index:0,count:3314462,average:43,stdev:0 GSM1810798_r1 1.51 6.95 0.3 91478233 108614812 71600785 89871852 118.73 125.52 0 0 0 0 0 0 57.3 74.54 3965299 1281466 3965299 1281466 62.28 71.68 3965299 1392876 3965299 1232379 14325765 15.66 6.37 0 15.61 0 1.16 0 0.47 0 0.00 0 30.90 0 2236584 0 43 0 41.65 0 1.41 0 0.00 0 1.20 0 0.01 0 331.45 0 0.30 0 211285 0 3314462 0 517345 0 38416 0 15437 0 0 0 1024025 0 20 0 0 0 667 0 47720 0 1333 0 49740 0 51.87 0 1719239 0 4208 56020 13.312737642586 3314462.0 2236584.0 211285.0 517345.0 38416.0 15437.0 0.0 1024025.0 1719239.0 67.5 6.4 15.6 1.2 0.5 0.0 30.9 51.9 43 43 43.00 38 142521866 26.4 21.7 21.8 30.1 0.0 35.9 24.6 smartseq 1062473 SRR2088364 SRP060416 SRS980134 SRX1082333 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810799: T75_P4_B1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810799 GSM1810799 T75_P4_B1_ILC1 169735663 3947341 2016-01-28 01:00:06 184986565 169735663 3947341 1 3947341 index:0,count:3947341,average:43,stdev:0 GSM1810799_r1 1.11 4.83 0.21 117844005 139620287 88006325 114737295 118.48 130.37 0 0 0 0 0 0 55.24 74.95 6035414 1576950 6035414 1576950 60.89 71.49 6035414 1738402 6035414 1504128 15174909 12.88 5.39 0 19.02 0 1.10 0 0.41 0 0.00 0 26.17 0 2854845 0 43 0 41.83 0 1.62 0 0.00 0 1.16 0 0.00 0 82.14 0 0.29 0 212683 0 3947341 0 750939 0 43395 0 16012 0 0 0 1033089 0 40 0 0 0 744 0 67717 0 1387 0 69888 0 53.30 0 2103906 0 6418 79587 12.400592084762 3947341.0 2854845.0 212683.0 750939.0 43395.0 16012.0 0.0 1033089.0 2103906.0 72.3 5.4 19.0 1.1 0.4 0.0 26.2 53.3 43 43 43.00 38 169735663 26.0 22.3 22.4 29.3 0.0 36.1 25.0 smartseq 1062490 SRR2088365 SRP060416 SRS979776 SRX1082334 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810800: T75_P4_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810800 GSM1810800 T75_P4_B4_NK 97220076 2260932 2016-01-28 01:00:06 106770138 97220076 2260932 1 2260932 index:0,count:2260932,average:43,stdev:0 GSM1810800_r1 2.38 6.24 0.24 64917111 77988983 51434960 65686586 120.14 127.71 0 0 0 0 0 0 56.34 72.27 2793214 890267 2793214 890267 60.61 69.03 2793214 957645 2793214 850472 10587033 16.31 5.84 0 15.40 0 1.12 0 0.45 0 0.00 0 28.54 0 1580089 0 43 0 41.75 0 1.46 0 0.00 0 1.18 0 0.00 0 280.67 0 0.28 0 131930 0 2260932 0 348144 0 25287 0 10220 0 0 0 645336 0 21 0 0 0 419 0 35054 0 850 0 36344 0 54.49 0 1231945 0 5069 40678 8.024856973762 2260932.0 1580089.0 131930.0 348144.0 25287.0 10220.0 0.0 645336.0 1231945.0 69.9 5.8 15.4 1.1 0.5 0.0 28.5 54.5 43 43 43.00 38 97220076 26.4 21.8 21.8 30.0 0.0 36.1 25.0 smartseq 1062506 SRR2088366 SRP060416 SRS980133 SRX1082335 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810801: T75_P4_B5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810801 GSM1810801 T75_P4_B5_ILC1 163638736 3805552 2016-01-28 01:00:06 178309870 163638736 3805552 1 3805552 index:0,count:3805552,average:43,stdev:0 GSM1810801_r1 2.15 4.28 0.14 118532973 139808616 88212166 114023281 117.95 129.26 0 0 0 0 0 0 53.98 73.38 6104793 1543906 6104793 1543906 60.3 69.91 6104793 1724826 6104793 1470819 16492774 13.91 4.77 0 19.87 0 1.13 0 0.45 0 0.00 0 23.26 0 2860271 0 43 0 41.93 0 1.41 0 0.00 0 1.14 0 0.00 0 489.29 0 0.28 0 181548 0 3805552 0 756289 0 43124 0 17107 0 0 0 885050 0 31 0 0 0 805 0 64183 0 1273 0 66292 0 55.29 0 2103982 0 6940 76812 11.068011527378 3805552.0 2860271.0 181548.0 756289.0 43124.0 17107.0 0.0 885050.0 2103982.0 75.2 4.8 19.9 1.1 0.4 0.0 23.3 55.3 43 43 43.00 38 163638736 26.3 22.2 22.3 29.2 0.0 36.1 25.3 smartseq 1062520 SRR2088367 SRP060416 SRS980132 SRX1082336 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810802: T75_P4_B8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810802 GSM1810802 T75_P4_B8_ILC1 37998627 883689 2016-01-28 01:00:06 43597655 37998627 883689 1 883689 index:0,count:883689,average:43,stdev:0 GSM1810802_r1 3.52 3.57 0.23 32220048 40017061 24636648 32550727 124.2 132.12 0 0 0 0 0 0 60.46 79.64 1493816 467105 1493816 467105 68.31 75.84 1493816 527817 1493816 444777 3141237 9.75 2.42 0 21.06 0 1.06 0 0.51 0 0.00 0 10.99 0 772632 0 43 0 42.01 0 1.37 0 0.00 0 1.13 0 0.00 0 227.23 0 0.33 0 21366 0 883689 0 186142 0 9354 0 4550 0 0 0 97153 0 12 0 0 0 179 0 19364 0 238 0 19793 0 66.37 0 586490 0 5736 23286 4.059623430962 883689.0 772632.0 21366.0 186142.0 9354.0 4550.0 0.0 97153.0 586490.0 87.4 2.4 21.1 1.1 0.5 0.0 11.0 66.4 43 43 43.00 38 37998627 27.4 21.5 22.5 28.7 0.0 35.3 24.4 smartseq 1062536 SRR2088368 SRP060416 SRS980131 SRX1082337 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810803: T75_P4_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810803 GSM1810803 T75_P4_B9_NK 26277902 611114 2016-01-28 01:00:06 29179052 26277902 611114 1 611114 index:0,count:611114,average:43,stdev:0 GSM1810803_r1 1.63 5.56 0.21 18493775 22039816 14486639 18380861 119.17 126.88 0 0 0 0 0 0 56.18 72.76 818019 252656 818019 252656 61.07 69.17 818019 274677 818019 240172 2802064 15.15 5.10 0 16.78 0 1.14 0 0.48 0 0.00 0 24.79 0 449754 0 43 0 41.72 0 1.41 0 0.00 0 1.11 0 0.00 0 146.67 0 0.28 0 31191 0 611114 0 102522 0 6947 0 2906 0 0 0 151507 0 2 0 0 0 156 0 11354 0 226 0 11738 0 56.82 0 347232 0 4178 13383 3.203207276209 611114.0 449754.0 31191.0 102522.0 6947.0 2906.0 0.0 151507.0 347232.0 73.6 5.1 16.8 1.1 0.5 0.0 24.8 56.8 43 43 43.00 38 26277902 26.7 21.7 21.7 29.8 0.0 36.1 24.9 smartseq 1062553 SRR2088369 SRP060416 SRS980130 SRX1082338 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810804: T75_P4_C10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810804 GSM1810804 T75_P4_C10_ILC1 48495443 1127801 2016-01-28 01:00:06 54608350 48495443 1127801 1 1127801 index:0,count:1127801,average:43,stdev:0 GSM1810804_r1 1.47 6.94 0.2 28923982 33119925 20160510 25107604 114.51 124.54 0 0 0 0 0 0 49.54 72.56 1537134 353429 1537134 353429 57.31 69.11 1537134 408906 1537134 336586 4710116 16.28 7.19 0 20.07 0 1.26 0 0.31 0 0.00 0 35.16 0 713454 0 43 0 41.39 0 1.40 0 0.00 0 1.16 0 0.00 0 270.67 0 0.33 0 81056 0 1127801 0 226391 0 14224 0 3549 0 0 0 396574 0 14 0 0 0 147 0 15808 0 429 0 16398 0 43.19 0 487063 0 3896 19099 4.902207392197 1127801.0 713454.0 81056.0 226391.0 14224.0 3549.0 0.0 396574.0 487063.0 63.3 7.2 20.1 1.3 0.3 0.0 35.2 43.2 43 43 43.00 38 48495443 25.9 21.4 21.7 31.0 0.0 35.6 23.9 smartseq 1062664 SRR2088370 SRP060416 SRS980129 SRX1082339 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810805: T75_P4_C11_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810805 GSM1810805 T75_P4_C11_ILC1 150290848 3495136 2016-01-28 01:00:06 163808806 150290848 3495136 1 3495136 index:0,count:3495136,average:43,stdev:0 GSM1810805_r1 3.69 5.33 0.25 107948849 134407579 83968428 110609562 124.51 131.73 0 0 0 0 0 0 57.8 75.31 4692939 1511927 4692939 1511927 64.58 72.31 4692939 1689282 4692939 1451717 14311978 13.26 4.79 0 17.40 0 1.11 0 0.50 0 0.00 0 23.54 0 2615888 0 43 0 41.82 0 1.38 0 0.00 0 1.15 0 0.00 0 292.62 0 0.28 0 167381 0 3495136 0 608194 0 38851 0 17529 0 0 0 822868 0 46 0 0 0 638 0 58824 0 1142 0 60650 0 57.44 0 2007694 0 5680 71490 12.586267605634 3495136.0 2615888.0 167381.0 608194.0 38851.0 17529.0 0.0 822868.0 2007694.0 74.8 4.8 17.4 1.1 0.5 0.0 23.5 57.4 43 43 43.00 38 150290848 26.7 21.7 21.8 29.8 0.0 36.2 25.2 smartseq 1062681 SRR2088371 SRP060416 SRS980128 SRX1082340 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810806: T75_P4_C12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810806 GSM1810806 T75_P4_C12_ILC1 144928662 3370434 2016-01-28 01:00:06 159128307 144928662 3370434 1 3370434 index:0,count:3370434,average:43,stdev:0 GSM1810806_r1 1.98 5.07 0.17 106779829 131670663 80254005 105669057 123.31 131.67 0 0 0 0 0 0 58.44 78.67 5007701 1510242 5007701 1510242 66.59 75.12 5007701 1720916 5007701 1442104 12247398 11.47 4.44 0 19.72 0 1.15 0 0.46 0 0.00 0 21.71 0 2584387 0 43 0 41.81 0 1.32 0 0.00 0 1.22 0 0.00 0 356.87 0 0.29 0 149629 0 3370434 0 664679 0 38654 0 15637 0 0 0 731756 0 12 0 0 0 637 0 70536 0 1185 0 72370 0 56.96 0 1919708 0 6794 86958 12.799234618781 3370434.0 2584387.0 149629.0 664679.0 38654.0 15637.0 0.0 731756.0 1919708.0 76.7 4.4 19.7 1.1 0.5 0.0 21.7 57.0 43 43 43.00 38 144928662 26.4 22.0 22.1 29.4 0.0 36.0 25.1 smartseq 1062697 SRR2088372 SRP060416 SRS980126 SRX1082341 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810807: T75_P4_C1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810807 GSM1810807 T75_P4_C1_ILC1 170957551 3975757 2016-01-28 01:00:06 185925356 170957551 3975757 1 3975757 index:0,count:3975757,average:43,stdev:0 GSM1810807_r1 2.93 4.45 0.17 121346688 145972103 85510342 112881808 120.29 132.01 0 0 0 0 0 0 53.59 76.95 6576090 1573040 6576090 1573040 62.86 73.36 6576090 1844919 6576090 1499682 15182997 12.51 5.03 0 22.41 0 1.16 0 0.34 0 0.00 0 24.68 0 2935077 0 43 0 41.83 0 1.42 0 0.00 0 1.11 0 0.00 0 280.64 0 0.28 0 200008 0 3975757 0 890799 0 45949 0 13554 0 0 0 981177 0 28 0 0 0 645 0 71320 0 1393 0 73386 0 51.42 0 2044278 0 6815 87933 12.902861335290 3975757.0 2935077.0 200008.0 890799.0 45949.0 13554.0 0.0 981177.0 2044278.0 73.8 5.0 22.4 1.2 0.3 0.0 24.7 51.4 43 43 43.00 38 170957551 26.0 22.4 22.5 29.2 0.0 36.2 25.2 smartseq 1062713 SRR2088373 SRP060416 SRS980127 SRX1082342 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810808: T75_P4_C3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810808 GSM1810808 T75_P4_C3_ILC1 117387850 2729950 2016-01-28 01:00:06 128469016 117387850 2729950 1 2729950 index:0,count:2729950,average:43,stdev:0 GSM1810808_r1 2.47 6.02 0.19 79020140 92837282 60071742 75324538 117.49 125.39 0 0 0 0 0 0 51.95 69.39 3733693 998734 3733693 998734 57.57 65.93 3733693 1106817 3733693 948954 13674989 17.31 5.71 0 17.70 0 1.19 0 0.52 0 0.00 0 27.88 0 1922434 0 43 0 41.74 0 1.30 0 0.00 0 1.12 0 0.00 0 280.79 0 0.29 0 155769 0 2729950 0 483130 0 32420 0 14095 0 0 0 761001 0 33 0 0 0 456 0 40555 0 1018 0 42062 0 52.72 0 1439304 0 5675 48497 8.545726872247 2729950.0 1922434.0 155769.0 483130.0 32420.0 14095.0 0.0 761001.0 1439304.0 70.4 5.7 17.7 1.2 0.5 0.0 27.9 52.7 43 43 43.00 38 117387850 26.3 21.9 21.9 29.9 0.0 36.1 25.0 smartseq 1062729 SRR2088374 SRP060416 SRS980125 SRX1082343 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810809: T75_P4_C4_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810809 GSM1810809 T75_P4_C4_ILC1 128733572 2993804 2016-01-28 01:00:06 140838700 128733572 2993804 1 2993804 index:0,count:2993804,average:43,stdev:0 GSM1810809_r1 1.83 4.88 0.19 95321501 117426843 72661257 96295821 123.19 132.53 0 0 0 0 0 0 57.76 76.58 4540843 1328145 4540843 1328145 63.9 72.31 4540843 1469436 4540843 1253995 12054385 12.65 4.43 0 18.88 0 1.10 0 0.51 0 0.00 0 21.59 0 2299475 0 43 0 41.90 0 1.38 0 0.00 0 1.12 0 0.00 0 299.38 0 0.28 0 132630 0 2993804 0 565231 0 32848 0 15174 0 0 0 646307 0 28 0 0 0 642 0 63613 0 1018 0 65301 0 57.93 0 1734244 0 7604 74689 9.822330352446 2993804.0 2299475.0 132630.0 565231.0 32848.0 15174.0 0.0 646307.0 1734244.0 76.8 4.4 18.9 1.1 0.5 0.0 21.6 57.9 43 43 43.00 38 128733572 26.1 22.4 22.4 29.1 0.0 36.2 25.4 smartseq 1062745 SRR2088375 SRP060416 SRS980124 SRX1082344 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810810: T75_P4_C5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810810 GSM1810810 T75_P4_C5_ILC1 152719445 3551615 2016-01-28 01:00:06 167765819 152719445 3551615 1 3551615 index:0,count:3551615,average:43,stdev:0 GSM1810810_r1 1.83 4.67 0.18 115331535 140408390 84855155 113066727 121.74 133.25 0 0 0 0 0 0 56.33 77.33 5911242 1565152 5911242 1565152 63.64 72.97 5911242 1768298 5911242 1476837 13671669 11.85 4.18 0 21.24 0 1.09 0 0.38 0 0.00 0 20.31 0 2778477 0 43 0 41.92 0 1.34 0 0.00 0 1.12 0 0.00 0 355.16 0 0.29 0 148607 0 3551615 0 754477 0 38596 0 13385 0 0 0 721157 0 26 0 0 0 623 0 75539 0 1093 0 77281 0 56.99 0 2024000 0 8593 90153 10.491446526242 3551615.0 2778477.0 148607.0 754477.0 38596.0 13385.0 0.0 721157.0 2024000.0 78.2 4.2 21.2 1.1 0.4 0.0 20.3 57.0 43 43 43.00 38 152719445 26.0 22.6 22.8 28.6 0.0 36.0 25.2 smartseq 1063320 SRR2088399 SRP060416 SRS980099 SRX1082368 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810834: T75_P4_E9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810834 GSM1810834 T75_P4_E9_ILC1 31199854 725578 2016-01-28 01:00:06 34693917 31199854 725578 1 725578 index:0,count:725578,average:43,stdev:0 GSM1810834_r1 2.47 5.07 0.2 23716263 29414352 19148695 24863126 124.03 129.84 0 0 0 0 0 0 60.87 76.21 963161 348188 963161 348188 65.59 72.11 963161 375173 963161 329494 3058489 12.90 4.15 0 15.86 0 1.04 0 0.44 0 0.00 0 19.69 0 571992 0 43 0 41.91 0 1.39 0 0.00 0 1.10 0 0.00 0 163.26 0 0.28 0 30090 0 725578 0 115090 0 7529 0 3177 0 0 0 142880 0 7 0 0 0 137 0 15990 0 273 0 16407 0 62.97 0 456902 0 5685 18381 3.233245382586 725578.0 571992.0 30090.0 115090.0 7529.0 3177.0 0.0 142880.0 456902.0 78.8 4.1 15.9 1.0 0.4 0.0 19.7 63.0 43 43 43.00 38 31199854 26.4 22.2 22.3 29.1 0.0 36.1 25.3 smartseq 1064968 SRR2088400 SRP060416 SRS980102 SRX1082369 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810835: T75_P4_F10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810835 GSM1810835 T75_P4_F10_ILC1 60724858 1412206 2016-01-28 01:00:06 67190931 60724858 1412206 1 1412206 index:0,count:1412206,average:43,stdev:0 GSM1810835_r1 2.25 5.32 0.22 45483124 55521285 35091580 45511826 122.07 129.69 0 0 0 0 0 0 58.12 76.18 2062951 637994 2062951 637994 64.61 72.65 2062951 709152 2062951 608423 5916538 13.01 4.28 0 18.42 0 1.04 0 0.42 0 0.00 0 20.81 0 1097668 0 43 0 41.90 0 1.42 0 0.00 0 1.11 0 0.00 0 317.75 0 0.28 0 60424 0 1412206 0 260177 0 14718 0 6000 0 0 0 293820 0 8 0 0 0 307 0 30576 0 473 0 31364 0 59.30 0 837491 0 6336 36115 5.699968434343 1412206.0 1097668.0 60424.0 260177.0 14718.0 6000.0 0.0 293820.0 837491.0 77.7 4.3 18.4 1.0 0.4 0.0 20.8 59.3 43 43 43.00 38 60724858 26.4 22.1 22.2 29.4 0.0 36.1 25.3 smartseq 1064985 SRR2088401 SRP060416 SRS980100 SRX1082370 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810836: T75_P4_F11_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810836 GSM1810836 T75_P4_F11_ILC1 182043381 4233567 2016-01-28 01:00:06 197618905 182043381 4233567 1 4233567 index:0,count:4233567,average:43,stdev:0 GSM1810836_r1 2.71 5.22 0.2 143087395 180237408 116242906 153099082 125.96 131.71 0 0 0 0 0 0 60.36 74.99 5666814 2076050 5666814 2076050 65.86 71.61 5666814 2265229 5666814 1982625 18355696 12.83 3.55 0 15.85 0 1.03 0 0.53 0 0.00 0 17.19 0 3439577 0 43 0 41.99 0 1.47 0 0.00 0 1.12 0 0.00 0 401.07 0 0.27 0 150218 0 4233567 0 671046 0 43639 0 22593 0 0 0 727758 0 39 0 0 0 669 0 95718 0 1582 0 98008 0 65.39 0 2768531 0 8453 111332 13.170708624157 4233567.0 3439577.0 150218.0 671046.0 43639.0 22593.0 0.0 727758.0 2768531.0 81.2 3.5 15.9 1.0 0.5 0.0 17.2 65.4 43 43 43.00 38 182043381 26.5 22.2 22.2 29.0 0.0 36.2 25.6 smartseq 1065001 SRR2088402 SRP060416 SRS980098 SRX1082371 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810837: T75_P4_F12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810837 GSM1810837 T75_P4_F12_ILC1 123366527 2868989 2016-01-28 01:00:06 134867600 123366527 2868989 1 2868989 index:0,count:2868989,average:43,stdev:0 GSM1810837_r1 2.69 5.44 0.22 94310561 118824023 76603602 100796456 125.99 131.58 0 0 0 0 0 0 62.07 77.21 3740559 1408024 3740559 1408024 67.61 74.04 3740559 1533634 3740559 1350211 11116751 11.79 4.00 0 15.49 0 1.07 0 0.47 0 0.00 0 19.40 0 2268264 0 43 0 42.00 0 1.52 0 0.00 0 1.13 0 0.00 0 90.60 0 0.27 0 114828 0 2868989 0 444534 0 30818 0 13453 0 0 0 556454 0 6 0 0 0 485 0 61931 0 895 0 63317 0 63.57 0 1823730 0 6503 74339 11.431493157004 2868989.0 2268264.0 114828.0 444534.0 30818.0 13453.0 0.0 556454.0 1823730.0 79.1 4.0 15.5 1.1 0.5 0.0 19.4 63.6 43 43 43.00 38 123366527 26.4 22.2 22.3 29.1 0.0 36.2 25.6 smartseq 1065018 SRR2088403 SRP060416 SRS980097 SRX1082372 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810838: T75_P4_F3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810838 GSM1810838 T75_P4_F3_ILC1 109715102 2551514 2016-01-28 01:00:06 120499578 109715102 2551514 1 2551514 index:0,count:2551514,average:43,stdev:0 GSM1810838_r1 2.0 4.06 0.22 81557262 97309655 60006999 78415554 119.31 130.68 0 0 0 0 0 0 54.82 75.3 4211239 1078066 4211239 1078066 62.11 71.53 4211239 1221255 4211239 1024116 10222858 12.53 4.45 0 20.96 0 1.15 0 0.48 0 0.00 0 21.30 0 1966431 0 43 0 41.91 0 1.26 0 0.00 0 1.14 0 0.00 0 306.18 0 0.28 0 113426 0 2551514 0 534750 0 29338 0 12294 0 0 0 543451 0 30 0 0 0 564 0 47890 0 857 0 49341 0 56.11 0 1431681 0 7700 59285 7.699350649351 2551514.0 1966431.0 113426.0 534750.0 29338.0 12294.0 0.0 543451.0 1431681.0 77.1 4.4 21.0 1.1 0.5 0.0 21.3 56.1 43 43 43.00 38 109715102 26.2 22.3 22.4 29.0 0.0 36.1 25.3 smartseq 1065035 SRR2088404 SRP060416 SRS980096 SRX1082373 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810839: T75_P4_F5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810839 GSM1810839 T75_P4_F5_ILC1 147048261 3419727 2016-01-28 01:00:06 160610371 147048261 3419727 1 3419727 index:0,count:3419727,average:43,stdev:0 GSM1810839_r1 2.09 3.87 0.16 112666742 136780755 82480999 110012469 121.4 133.38 0 0 0 0 0 0 54.72 75.43 5947269 1482731 5947269 1482731 62.42 71.96 5947269 1691538 5947269 1414516 13893523 12.33 4.00 0 21.76 0 1.16 0 0.41 0 0.00 0 19.19 0 2709882 0 43 0 41.96 0 1.42 0 0.00 0 1.11 0 0.00 0 362.09 0 0.28 0 136744 0 3419727 0 744283 0 39546 0 13993 0 0 0 656306 0 13 0 0 0 524 0 66676 0 949 0 68162 0 57.48 0 1965599 0 7990 79242 9.917647058824 3419727.0 2709882.0 136744.0 744283.0 39546.0 13993.0 0.0 656306.0 1965599.0 79.2 4.0 21.8 1.2 0.4 0.0 19.2 57.5 43 43 43.00 38 147048261 26.4 22.2 22.3 29.1 0.0 36.2 25.5 smartseq 1065050 SRR2088405 SRP060416 SRS980095 SRX1082374 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810840: T75_P4_F6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810840 GSM1810840 T75_P4_F6_ILC1 118594430 2758010 2016-01-28 01:00:06 130239984 118594430 2758010 1 2758010 index:0,count:2758010,average:43,stdev:0 GSM1810840_r1 2.38 5.27 0.25 84546060 102022692 64678290 83651696 120.67 129.34 0 0 0 0 0 0 54.22 71.84 3946877 1110658 3946877 1110658 60.62 68.97 3946877 1241865 3946877 1066259 12764721 15.10 4.92 0 18.22 0 1.23 0 0.46 0 0.00 0 24.03 0 2048566 0 43 0 41.84 0 1.38 0 0.00 0 1.16 0 0.01 0 292.02 0 0.28 0 135738 0 2758010 0 502600 0 34049 0 12748 0 0 0 662647 0 20 0 0 0 530 0 45255 0 977 0 46782 0 56.05 0 1545966 0 5865 55895 9.530264279625 2758010.0 2048566.0 135738.0 502600.0 34049.0 12748.0 0.0 662647.0 1545966.0 74.3 4.9 18.2 1.2 0.5 0.0 24.0 56.1 43 43 43.00 38 118594430 26.5 21.8 21.8 29.9 0.0 36.1 25.0 smartseq 1065066 SRR2088406 SRP060416 SRS980094 SRX1082375 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810841: T75_P4_F7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810841 GSM1810841 T75_P4_F7_ILC1 144126798 3351786 2016-01-28 01:00:06 157588335 144126798 3351786 1 3351786 index:0,count:3351786,average:43,stdev:0 GSM1810841_r1 1.63 3.65 0.17 118955676 147246325 89945007 120703528 123.78 134.2 0 0 0 0 0 0 59.69 79.45 5825301 1700506 5825301 1700506 67.36 75.53 5825301 1918935 5825301 1616484 12015296 10.10 2.83 0 21.14 0 1.18 0 0.52 0 0.00 0 13.30 0 2848940 0 43 0 42.02 0 1.44 0 0.00 0 1.15 0 0.00 0 335.18 0 0.27 0 94726 0 3351786 0 708638 0 39670 0 17532 0 0 0 445644 0 12 0 0 0 824 0 88089 0 986 0 89911 0 63.86 0 2140302 0 10707 106618 9.957784626880 3351786.0 2848940.0 94726.0 708638.0 39670.0 17532.0 0.0 445644.0 2140302.0 85.0 2.8 21.1 1.2 0.5 0.0 13.3 63.9 43 43 43.00 38 144126798 26.2 22.7 22.8 28.2 0.0 36.2 25.7 smartseq 1065082 SRR2088407 SRP060416 SRS980092 SRX1082376 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810842: T75_P4_F8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810842 GSM1810842 T75_P4_F8_ILC1 99086319 2304333 2016-01-28 01:00:06 109030744 99086319 2304333 1 2304333 index:0,count:2304333,average:43,stdev:0 GSM1810842_r1 1.9 3.52 0.16 85037551 104908725 63014080 83849272 123.37 133.06 0 0 0 0 0 0 58.69 79.67 4275451 1194424 4275451 1194424 67.47 75.41 4275451 1373176 4275451 1130574 8643137 10.16 2.25 0 23.26 0 1.03 0 0.32 0 0.00 0 10.34 0 2035188 0 43 0 42.03 0 1.33 0 0.00 0 1.14 0 0.00 0 360.68 0 0.28 0 51855 0 2304333 0 535960 0 23634 0 7357 0 0 0 238154 0 8 0 0 0 630 0 57533 0 786 0 58957 0 65.06 0 1499228 0 7111 70710 9.943749121080 2304333.0 2035188.0 51855.0 535960.0 23634.0 7357.0 0.0 238154.0 1499228.0 88.3 2.3 23.3 1.0 0.3 0.0 10.3 65.1 43 43 43.00 38 99086319 26.6 22.5 22.7 28.2 0.0 36.2 25.8 smartseq 1065098 SRR2088408 SRP060416 SRS980093 SRX1082377 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810843: T75_P4_F9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810843 GSM1810843 T75_P4_F9_ILC1 19357654 450178 2016-01-28 01:00:06 21534514 19357654 450178 1 450178 index:0,count:450178,average:43,stdev:0 GSM1810843_r1 1.42 5.02 0.17 14336569 17453387 10866728 14216402 121.74 130.83 0 0 0 0 0 0 58.44 78.04 683552 202878 683552 202878 64.93 74.24 683552 225399 683552 193002 1715332 11.96 4.51 0 19.36 0 1.05 0 0.37 0 0.00 0 21.46 0 347142 0 43 0 41.80 0 1.45 0 0.00 0 1.21 0 0.01 0 115.76 0 0.29 0 20295 0 450178 0 87173 0 4742 0 1669 0 0 0 96625 0 0 0 0 0 119 0 9665 0 162 0 9946 0 57.75 0 259969 0 4344 11406 2.625690607735 450178.0 347142.0 20295.0 87173.0 4742.0 1669.0 0.0 96625.0 259969.0 77.1 4.5 19.4 1.1 0.4 0.0 21.5 57.7 43 43 43.00 38 19357654 26.6 21.9 21.9 29.6 0.0 36.1 25.1 smartseq 1065112 SRR2088409 SRP060416 SRS980091 SRX1082378 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810844: T75_P4_G10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810844 GSM1810844 T75_P4_G10_ILC1 55660404 1294428 2016-01-28 01:00:06 61402450 55660404 1294428 1 1294428 index:0,count:1294428,average:43,stdev:0 GSM1810844_r1 2.09 4.62 0.18 42251491 52135830 31931925 42440014 123.39 132.91 0 0 0 0 0 0 59.71 79.84 2002239 608969 2002239 608969 67.27 76.49 2002239 686110 2002239 583406 4718818 11.17 4.06 0 19.87 0 1.03 0 0.41 0 0.00 0 19.76 0 1019927 0 43 0 41.86 0 1.36 0 0.00 0 1.14 0 0.00 0 245.26 0 0.28 0 52498 0 1294428 0 257172 0 13358 0 5354 0 0 0 255789 0 5 0 0 0 223 0 27312 0 442 0 27982 0 58.93 0 762755 0 6365 33252 5.224194815397 1294428.0 1019927.0 52498.0 257172.0 13358.0 5354.0 0.0 255789.0 762755.0 78.8 4.1 19.9 1.0 0.4 0.0 19.8 58.9 43 43 43.00 38 55660404 26.4 22.1 22.2 29.3 0.0 36.2 25.4 smartseq 1065288 SRR2088414 SRP060416 SRS980086 SRX1082383 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810849: T75_P4_G6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810849 GSM1810849 T75_P4_G6_ILC1 127097938 2955766 2016-01-28 01:00:06 139532814 127097938 2955766 1 2955766 index:0,count:2955766,average:43,stdev:0 GSM1810849_r1 2.67 4.92 0.24 89733249 107168272 66742135 86719764 119.43 129.93 0 0 0 0 0 0 54.44 74.15 4504142 1184382 4504142 1184382 60.83 70.52 4504142 1323493 4504142 1126430 12291432 13.70 5.12 0 19.57 0 1.16 0 0.50 0 0.00 0 24.74 0 2175627 0 43 0 41.79 0 1.34 0 0.00 0 1.16 0 0.01 0 287.59 0 0.29 0 151399 0 2955766 0 578393 0 34194 0 14835 0 0 0 731110 0 18 0 0 0 629 0 50256 0 1048 0 51951 0 54.04 0 1597234 0 6360 60552 9.520754716981 2955766.0 2175627.0 151399.0 578393.0 34194.0 14835.0 0.0 731110.0 1597234.0 73.6 5.1 19.6 1.2 0.5 0.0 24.7 54.0 43 43 43.00 38 127097938 26.2 22.1 22.2 29.5 0.0 36.1 25.0 smartseq 1065304 SRR2088415 SRP060416 SRS980085 SRX1082384 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810850: T75_P4_G7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810850 GSM1810850 T75_P4_G7_ILC1 94947698 2208086 2016-01-28 01:00:06 104841190 94947698 2208086 1 2208086 index:0,count:2208086,average:43,stdev:0 GSM1810850_r1 1.45 4.8 0.19 70880775 84465156 53873214 69399874 119.17 128.82 0 0 0 0 0 0 56.75 75.55 3436544 974082 3436544 974082 62.82 71.97 3436544 1078210 3436544 928001 8965694 12.65 4.30 0 19.34 0 1.11 0 0.46 0 0.00 0 20.70 0 1716483 0 43 0 41.78 0 1.45 0 0.00 0 1.17 0 0.01 0 441.62 0 0.29 0 94885 0 2208086 0 427135 0 24528 0 10088 0 0 0 456987 0 12 0 0 0 498 0 46137 0 783 0 47430 0 58.39 0 1289348 0 7195 55312 7.687560806115 2208086.0 1716483.0 94885.0 427135.0 24528.0 10088.0 0.0 456987.0 1289348.0 77.7 4.3 19.3 1.1 0.5 0.0 20.7 58.4 43 43 43.00 38 94947698 26.5 22.0 22.1 29.5 0.0 36.0 25.0 smartseq 1065352 SRR2088418 SRP060416 SRS980081 SRX1082387 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810853: T75_P4_H12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810853 GSM1810853 T75_P4_H12_ILC1 145265008 3378256 2016-01-28 01:00:06 159740895 145265008 3378256 1 3378256 index:0,count:3378256,average:43,stdev:0 GSM1810853_r1 1.18 5.09 0.24 105618644 124893309 80777463 102398993 118.25 126.77 0 0 0 0 0 0 55.81 73.88 4952145 1426595 4952145 1426595 61.96 70.49 4952145 1583858 4952145 1361071 14756509 13.97 4.67 0 18.52 0 1.13 0 0.51 0 0.00 0 22.70 0 2556319 0 43 0 41.84 0 1.32 0 0.00 0 1.16 0 0.00 0 304.04 0 0.29 0 157640 0 3378256 0 625487 0 38059 0 17121 0 0 0 766757 0 11 0 0 0 606 0 62110 0 1243 0 63970 0 57.15 0 1930832 0 6802 76404 11.232578653337 3378256.0 2556319.0 157640.0 625487.0 38059.0 17121.0 0.0 766757.0 1930832.0 75.7 4.7 18.5 1.1 0.5 0.0 22.7 57.2 43 43 43.00 38 145265008 26.5 22.0 22.0 29.6 0.0 36.0 24.9 smartseq 1065368 SRR2088419 SRP060416 SRS980082 SRX1082388 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810854: T75_P4_H3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810854 GSM1810854 T75_P4_H3_ILC1 101835739 2368273 2016-01-28 01:00:06 112903344 101835739 2368273 1 2368273 index:0,count:2368273,average:43,stdev:0 GSM1810854_r1 1.41 5.68 0.27 71550014 85765582 55940509 71597902 119.87 127.99 0 0 0 0 0 0 58.16 75.47 3179973 1010781 3179973 1010781 63.08 71.87 3179973 1096216 3179973 962507 9565923 13.37 5.20 0 16.83 0 1.04 0 0.42 0 0.00 0 25.16 0 1737845 0 43 0 41.77 0 1.48 0 0.00 0 1.14 0 0.00 0 275.03 0 0.30 0 123098 0 2368273 0 398547 0 24746 0 9906 0 0 0 595776 0 5 0 0 0 473 0 47294 0 980 0 48752 0 56.55 0 1339298 0 6514 54767 8.407583665950 2368273.0 1737845.0 123098.0 398547.0 24746.0 9906.0 0.0 595776.0 1339298.0 73.4 5.2 16.8 1.0 0.4 0.0 25.2 56.6 43 43 43.00 38 101835739 26.3 21.9 22.0 29.8 0.0 35.9 24.7 smartseq 1065480 SRR2088420 SRP060416 SRS980080 SRX1082389 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810855: T75_P4_H5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810855 GSM1810855 T75_P4_H5_ILC1 138129115 3212305 2016-01-28 01:00:06 151845865 138129115 3212305 1 3212305 index:0,count:3212305,average:43,stdev:0 GSM1810855_r1 0.78 5.12 0.23 95588300 108563442 72025781 89139431 113.57 123.76 0 0 0 0 0 0 51.76 69.67 4793282 1199838 4793282 1199838 57.56 66.96 4793282 1334331 4793282 1153042 14588730 15.26 5.35 0 18.56 0 1.21 0 0.50 0 0.00 0 26.13 0 2318200 0 43 0 41.83 0 1.32 0 0.00 0 1.12 0 0.00 0 66.08 0 0.30 0 171919 0 3212305 0 596136 0 38739 0 15935 0 0 0 839431 0 25 0 0 0 655 0 50308 0 1092 0 52080 0 53.61 0 1722064 0 6483 62966 9.712478790683 3212305.0 2318200.0 171919.0 596136.0 38739.0 15935.0 0.0 839431.0 1722064.0 72.2 5.4 18.6 1.2 0.5 0.0 26.1 53.6 43 43 43.00 38 138129115 26.5 21.8 21.9 29.8 0.0 36.0 24.8 smartseq 1065497 SRR2088421 SRP060416 SRS979778 SRX1082390 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810856: T75_P4_H8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810856 GSM1810856 T75_P4_H8_ILC1 60068248 1396936 2016-01-28 01:00:06 66943930 60068248 1396936 1 1396936 index:0,count:1396936,average:43,stdev:0 GSM1810856_r1 0.8 4.21 0.25 50132435 59769958 38591668 48929088 119.22 126.79 0 0 0 0 0 0 54.6 71.51 2365577 657315 2365577 657315 62.06 68.36 2365577 747014 2365577 628311 7321873 14.61 2.56 0 20.37 0 1.36 0 0.55 0 0.00 0 11.91 0 1203774 0 43 0 41.99 0 1.34 0 0.00 0 1.13 0 0.01 0 193.42 0 0.29 0 35827 0 1396936 0 284598 0 19061 0 7723 0 0 0 166378 0 3 0 0 0 355 0 28504 0 454 0 29316 0 65.80 0 919176 0 5182 35435 6.838093400232 1396936.0 1203774.0 35827.0 284598.0 19061.0 7723.0 0.0 166378.0 919176.0 86.2 2.6 20.4 1.4 0.6 0.0 11.9 65.8 43 43 43.00 38 60068248 27.3 21.7 21.9 29.1 0.0 36.0 25.3 smartseq 1065513 SRR2088422 SRP060416 SRS980079 SRX1082391 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810857: T75_P4_H9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810857 GSM1810857 T75_P4_H9_ILC1 18194160 423120 2016-01-28 01:00:06 20329375 18194160 423120 1 423120 index:0,count:423120,average:43,stdev:0 GSM1810857_r1 3.31 5.61 0.27 12378639 15032220 9489993 12408130 121.44 130.75 0 0 0 0 0 0 56.21 74.4 578721 169394 578721 169394 61.26 70.69 578721 184610 578721 160941 1755048 14.18 5.61 0 17.41 0 1.12 0 0.48 0 0.00 0 27.18 0 301344 0 43 0 41.68 0 1.31 0 0.00 0 1.19 0 0.00 0 117.17 0 0.29 0 23722 0 423120 0 73678 0 4759 0 2027 0 0 0 114990 0 4 0 0 0 98 0 7671 0 155 0 7928 0 53.81 0 227666 0 3392 8467 2.496167452830 423120.0 301344.0 23722.0 73678.0 4759.0 2027.0 0.0 114990.0 227666.0 71.2 5.6 17.4 1.1 0.5 0.0 27.2 53.8 43 43 43.00 38 18194160 26.6 21.7 21.7 30.0 0.0 36.0 24.8 smartseq 1065532 SRR2088423 SRP060416 SRS980078 SRX1082392 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810858: T86_P1_A10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810858 GSM1810858 T86_P1_A10_ILC3 29225595 679665 2016-01-28 01:00:06 33515397 29225595 679665 1 679665 index:0,count:679665,average:43,stdev:0 GSM1810858_r1 6.12 8.46 0.2 17593751 21542224 13512056 17228864 122.44 127.51 0 0 0 0 0 0 53.84 71.7 726001 233506 726001 233506 60.69 68.75 726001 263220 726001 223921 3007196 17.09 7.75 0 15.89 0 1.00 0 0.32 0 0.00 0 34.87 0 433685 0 43 0 41.49 0 1.14 0 0.01 0 1.15 0 0.00 0 122.34 0 0.46 0 52656 0 679665 0 107994 0 6799 0 2161 0 0 0 237020 0 7 0 0 0 85 0 9944 0 223 0 10259 0 47.92 0 325691 0 4713 11468 2.433269679610 679665.0 433685.0 52656.0 107994.0 6799.0 2161.0 0.0 237020.0 325691.0 63.8 7.7 15.9 1.0 0.3 0.0 34.9 47.9 43 43 43.00 38 29225595 27.0 21.1 22.0 29.8 0.0 34.5 22.4 smartseq 1065547 SRR2088424 SRP060416 SRS980077 SRX1082393 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810859: T86_P1_A12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810859 GSM1810859 T86_P1_A12_ILC3 44079988 1025116 2016-01-28 01:00:06 50542265 44079988 1025116 1 1025116 index:0,count:1025116,average:43,stdev:0 GSM1810859_r1 9.09 7.14 0.18 29664398 38739875 23789003 31966751 130.59 134.38 0 0 0 0 0 0 59.93 75.86 1114452 432404 1114452 432404 67.19 72.95 1114452 484789 1114452 415819 4015641 13.54 6.42 0 14.78 0 0.81 0 0.35 0 0.00 0 28.46 0 721538 0 43 0 41.73 0 1.17 0 0.01 0 1.12 0 0.01 0 205.02 0 0.46 0 65774 0 1025116 0 151510 0 8313 0 3568 0 0 0 291697 0 6 0 0 0 126 0 18652 0 292 0 19076 0 55.61 0 570028 0 7367 20746 2.816071670965 1025116.0 721538.0 65774.0 151510.0 8313.0 3568.0 0.0 291697.0 570028.0 70.4 6.4 14.8 0.8 0.3 0.0 28.5 55.6 43 43 43.00 38 44079988 26.8 21.5 22.6 29.1 0.0 34.5 22.7 smartseq 1065565 SRR2088425 SRP060416 SRS980076 SRX1082394 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810860: T86_P1_A1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810860 GSM1810860 T86_P1_A1_ILC3 56568736 1315552 2016-01-28 01:00:06 64379971 56568736 1315552 1 1315552 index:0,count:1315552,average:43,stdev:0 GSM1810860_r1 9.94 7.58 0.2 36445895 47783558 28244445 38401604 131.11 135.96 0 0 0 0 0 0 59.2 77.77 1437914 527366 1437914 527366 67.01 74.2 1437914 596946 1437914 503145 4923786 13.51 6.99 0 16.17 0 0.87 0 0.26 0 0.00 0 31.17 0 890780 0 43 0 41.65 0 1.17 0 0.01 0 1.13 0 0.00 0 236.80 0 0.44 0 91945 0 1315552 0 212688 0 11389 0 3360 0 0 0 410023 0 17 0 0 0 159 0 23152 0 371 0 23699 0 51.54 0 678092 0 8527 26734 3.135217544271 1315552.0 890780.0 91945.0 212688.0 11389.0 3360.0 0.0 410023.0 678092.0 67.7 7.0 16.2 0.9 0.3 0.0 31.2 51.5 43 43 43.00 38 56568736 26.6 21.5 22.3 29.6 0.0 34.7 22.8 smartseq 1065580 SRR2088426 SRP060416 SRS980075 SRX1082395 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810861: T86_P1_A3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810861 GSM1810861 T86_P1_A3_ILC3 145573490 3385430 2016-01-28 01:00:06 158505441 145573490 3385430 1 3385430 index:0,count:3385430,average:43,stdev:0 GSM1810861_r1 6.22 5.55 0.19 72198001 90327845 57648670 74731695 125.11 129.63 0 0 0 0 0 0 51.16 64.84 2828566 889949 2828566 889949 57.67 61.94 2828566 1003177 2828566 850149 13129945 18.19 9.86 0 10.84 0 0.92 0 0.46 0 0.00 0 47.24 0 1739523 0 43 0 42.00 0 1.19 0 0.01 0 1.12 0 0.01 0 248.73 0 0.38 0 333679 0 3385430 0 367036 0 30988 0 15607 0 0 0 1599312 0 27 0 0 0 294 0 36589 0 977 0 37887 0 40.54 0 1372487 0 10182 42788 4.202317815753 3385430.0 1739523.0 333679.0 367036.0 30988.0 15607.0 0.0 1599312.0 1372487.0 51.4 9.9 10.8 0.9 0.5 0.0 47.2 40.5 43 43 43.00 38 145573490 27.7 21.2 21.6 29.4 0.0 35.9 24.9 smartseq 1065592 SRR2088427 SRP060416 SRS980074 SRX1082396 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810862: T86_P1_A5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810862 GSM1810862 T86_P1_A5_ILC3 58048022 1349954 2016-01-28 01:00:06 64964701 58048022 1349954 1 1349954 index:0,count:1349954,average:43,stdev:0 GSM1810862_r1 6.71 8.54 0.23 32727774 39810272 24372299 31124958 121.64 127.71 0 0 0 0 0 0 49.91 68.74 1435406 404658 1435406 404658 56.97 65.81 1435406 461940 1435406 387439 6255711 19.11 8.60 0 16.45 0 1.07 0 0.28 0 0.00 0 38.59 0 810795 0 43 0 41.40 0 1.15 0 0.01 0 1.17 0 0.01 0 242.99 0 0.43 0 116101 0 1349954 0 222086 0 14409 0 3776 0 0 0 520974 0 15 0 0 0 150 0 17032 0 459 0 17656 0 43.61 0 588709 0 4935 19113 3.872948328267 1349954.0 810795.0 116101.0 222086.0 14409.0 3776.0 0.0 520974.0 588709.0 60.1 8.6 16.5 1.1 0.3 0.0 38.6 43.6 43 43 43.00 38 58048022 26.9 21.4 21.8 29.9 0.0 35.1 23.0 smartseq 1065609 SRR2088428 SRP060416 SRS980073 SRX1082397 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810863: T86_P1_A8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810863 GSM1810863 T86_P1_A8_ILC3 57651175 1340725 2016-01-28 01:00:06 64504503 57651175 1340725 1 1340725 index:0,count:1340725,average:43,stdev:0 GSM1810863_r1 8.43 7.39 0.23 35987659 46292627 28356456 37687887 128.63 132.91 0 0 0 0 0 0 56.27 72.62 1400019 493127 1400019 493127 63.44 69.49 1400019 555950 1400019 471864 5581705 15.51 7.50 0 14.72 0 0.93 0 0.35 0 0.00 0 33.35 0 876404 0 43 0 41.76 0 1.18 0 0.01 0 1.13 0 0.01 0 268.14 0 0.39 0 100545 0 1340725 0 197356 0 12436 0 4696 0 0 0 447189 0 6 0 0 0 126 0 21126 0 452 0 21710 0 50.65 0 679048 0 7517 24358 3.240388452840 1340725.0 876404.0 100545.0 197356.0 12436.0 4696.0 0.0 447189.0 679048.0 65.4 7.5 14.7 0.9 0.4 0.0 33.4 50.6 43 43 43.00 38 57651175 26.5 22.0 22.3 29.1 0.0 35.2 23.4 smartseq 1065624 SRR2088429 SRP060416 SRS980072 SRX1082398 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810864: T86_P1_B12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810864 GSM1810864 T86_P1_B12_ILC3 144086163 3350841 2016-01-28 01:00:06 157030303 144086163 3350841 1 3350841 index:0,count:3350841,average:43,stdev:0 GSM1810864_r1 11.27 6.6 0.23 90312239 118835993 70225382 95902014 131.58 136.56 0 0 0 0 0 0 57.83 75.55 3601528 1271851 3601528 1271851 65.7 72.01 3601528 1444975 3601528 1212191 13386881 14.82 7.42 0 15.40 0 0.93 0 0.35 0 0.00 0 33.09 0 2199447 0 43 0 41.71 0 1.08 0 0.01 0 1.14 0 0.01 0 194.56 0 0.39 0 248633 0 3350841 0 515987 0 31117 0 11568 0 0 0 1108709 0 17 0 0 0 408 0 53863 0 977 0 55265 0 50.24 0 1683460 0 10697 63143 5.902869963541 3350841.0 2199447.0 248633.0 515987.0 31117.0 11568.0 0.0 1108709.0 1683460.0 65.6 7.4 15.4 0.9 0.3 0.0 33.1 50.2 43 43 43.00 38 144086163 26.3 22.3 22.6 28.8 0.0 35.7 24.0 smartseq 1065737 SRR2088430 SRP060416 SRS980069 SRX1082399 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810865: T86_P1_B1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810865 GSM1810865 T86_P1_B1_ILC3 130876219 3043633 2016-01-28 01:00:06 142143181 130876219 3043633 1 3043633 index:0,count:3043633,average:43,stdev:0 GSM1810865_r1 9.37 6.7 0.2 85406195 112259607 69045511 93203515 131.44 134.99 0 0 0 0 0 0 62.28 78.18 3181399 1290681 3181399 1290681 69.14 75.0 3181399 1432693 3181399 1238186 11351859 13.29 6.91 0 13.85 0 0.82 0 0.28 0 0.00 0 30.82 0 2072266 0 43 0 41.82 0 1.13 0 0.01 0 1.10 0 0.01 0 260.88 0 0.36 0 210406 0 3043633 0 421438 0 24966 0 8490 0 0 0 937911 0 3 0 0 0 390 0 57883 0 922 0 59198 0 54.24 0 1650828 0 10368 66391 6.403452932099 3043633.0 2072266.0 210406.0 421438.0 24966.0 8490.0 0.0 937911.0 1650828.0 68.1 6.9 13.8 0.8 0.3 0.0 30.8 54.2 43 43 43.00 38 130876219 26.4 22.2 22.5 28.9 0.0 35.8 24.2 smartseq 1065754 SRR2088431 SRP060416 SRS980071 SRX1082400 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810866: T86_P1_B3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810866 GSM1810866 T86_P1_B3_ILC3 139059850 3233950 2016-01-28 01:00:06 151114432 139059850 3233950 1 3233950 index:0,count:3233950,average:43,stdev:0 GSM1810866_r1 10.38 5.87 0.18 97175117 131253560 77963321 108302903 135.07 138.92 0 0 0 0 0 0 63.62 80.17 3628863 1490748 3628863 1490748 71.21 76.28 3628863 1668694 3628863 1418467 10810369 11.12 5.88 0 14.95 0 0.86 0 0.37 0 0.00 0 26.31 0 2343199 0 43 0 41.93 0 1.16 0 0.01 0 1.12 0 0.01 0 291.06 0 0.36 0 190001 0 3233950 0 483616 0 27848 0 12087 0 0 0 850816 0 53 0 0 0 568 0 70308 0 966 0 71895 0 57.50 0 1859583 0 13986 81753 5.845345345345 3233950.0 2343199.0 190001.0 483616.0 27848.0 12087.0 0.0 850816.0 1859583.0 72.5 5.9 15.0 0.9 0.4 0.0 26.3 57.5 43 43 43.00 38 139059850 26.3 22.7 22.9 28.1 0.0 35.8 24.4 smartseq 1065769 SRR2088432 SRP060416 SRS980070 SRX1082401 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810867: T86_P1_B5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810867 GSM1810867 T86_P1_B5_ILC3 134855998 3136186 2016-01-28 01:00:06 146567279 134855998 3136186 1 3136186 index:0,count:3136186,average:43,stdev:0 GSM1810867_r1 11.19 6.62 0.18 89588837 120880958 71721136 99630815 134.93 138.91 0 0 0 0 0 0 61.79 78.28 3364539 1343563 3364539 1343563 69.02 74.88 3364539 1500741 3364539 1285266 12139154 13.55 6.60 0 14.60 0 0.82 0 0.37 0 0.00 0 29.48 0 2174303 0 43 0 41.79 0 1.11 0 0.01 0 1.11 0 0.01 0 250.89 0 0.37 0 206902 0 3136186 0 457878 0 25720 0 11490 0 0 0 924673 0 26 0 0 0 484 0 56549 0 1005 0 58064 0 54.73 0 1716425 0 10787 65267 6.050523778622 3136186.0 2174303.0 206902.0 457878.0 25720.0 11490.0 0.0 924673.0 1716425.0 69.3 6.6 14.6 0.8 0.4 0.0 29.5 54.7 43 43 43.00 38 134855998 26.5 22.4 22.6 28.6 0.0 35.8 24.1 smartseq 1065786 SRR2088433 SRP060416 SRS980068 SRX1082402 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810868: T86_P1_B6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810868 GSM1810868 T86_P1_B6_ILC3 104852017 2438419 2016-01-28 01:00:06 115877627 104852017 2438419 1 2438419 index:0,count:2438419,average:43,stdev:0 GSM1810868_r1 7.77 8.46 0.22 58184667 73569805 44741269 59009368 126.44 131.89 0 0 0 0 0 0 53.23 70.84 2411980 763935 2411980 763935 60.02 68.27 2411980 861247 2411980 736204 10329478 17.75 8.88 0 14.63 0 1.13 0 0.29 0 0.00 0 39.72 0 1435047 0 43 0 41.49 0 1.17 0 0.01 0 1.12 0 0.01 0 292.61 0 0.40 0 216468 0 2438419 0 356668 0 27662 0 7191 0 0 0 968519 0 2 0 0 0 312 0 30253 0 755 0 31322 0 44.22 0 1078379 0 6363 34338 5.396511079679 2438419.0 1435047.0 216468.0 356668.0 27662.0 7191.0 0.0 968519.0 1078379.0 58.9 8.9 14.6 1.1 0.3 0.0 39.7 44.2 43 43 43.00 38 104852017 26.5 21.7 22.1 29.7 0.0 35.3 23.3 smartseq 1065801 SRR2088434 SRP060416 SRS980067 SRX1082403 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810869: T86_P1_B7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810869 GSM1810869 T86_P1_B7_ILC3 132819045 3088815 2016-01-28 01:00:06 144674296 132819045 3088815 1 3088815 index:0,count:3088815,average:43,stdev:0 GSM1810869_r1 7.7 7.64 0.24 74701649 95102833 57998974 76803925 127.31 132.42 0 0 0 0 0 0 57.18 75.14 3034348 1048191 3034348 1048191 64.23 72.01 3034348 1177284 3034348 1004554 10900164 14.59 8.79 0 14.18 0 1.03 0 0.35 0 0.00 0 39.28 0 1832996 0 43 0 41.58 0 1.10 0 0.01 0 1.16 0 0.01 0 205.92 0 0.39 0 271526 0 3088815 0 438023 0 31895 0 10726 0 0 0 1213198 0 49 0 0 0 322 0 39155 0 1056 0 40582 0 45.16 0 1394973 0 7010 46589 6.646077032810 3088815.0 1832996.0 271526.0 438023.0 31895.0 10726.0 0.0 1213198.0 1394973.0 59.3 8.8 14.2 1.0 0.3 0.0 39.3 45.2 43 43 43.00 38 132819045 26.2 22.4 22.8 28.6 0.0 35.6 23.8 smartseq 1065817 SRR2088435 SRP060416 SRS980066 SRX1082404 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810870: T86_P1_B8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810870 GSM1810870 T86_P1_B8_ILC3 57008282 1325774 2016-01-28 01:00:06 64653630 57008282 1325774 1 1325774 index:0,count:1325774,average:43,stdev:0 GSM1810870_r1 9.07 5.45 0.17 42193333 56005721 34186933 46541451 132.74 136.14 0 0 0 0 0 0 59.13 73.72 1531711 600732 1531711 600732 65.88 69.76 1531711 669316 1531711 568430 5870448 13.91 4.83 0 15.17 0 0.96 0 0.53 0 0.00 0 21.88 0 1015918 0 43 0 41.96 0 1.12 0 0.01 0 1.16 0 0.00 0 61.98 0 0.45 0 64053 0 1325774 0 201074 0 12712 0 7003 0 0 0 290141 0 18 0 0 0 222 0 25405 0 406 0 26051 0 61.46 0 814844 0 9797 29655 3.026947024599 1325774.0 1015918.0 64053.0 201074.0 12712.0 7003.0 0.0 290141.0 814844.0 76.6 4.8 15.2 1.0 0.5 0.0 21.9 61.5 43 43 43.00 38 57008282 26.9 22.1 23.0 28.0 0.0 34.9 23.2 smartseq 1065832 SRR2088436 SRP060416 SRS980065 SRX1082405 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810871: T86_P1_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810871 GSM1810871 T86_P1_B9_NK 30097248 699936 2016-01-28 01:00:06 33153549 30097248 699936 1 699936 index:0,count:699936,average:43,stdev:0 GSM1810871_r1 6.39 5.71 0.17 21332360 28561757 17301003 23775892 133.89 137.42 0 0 0 0 0 0 61.91 77.14 778213 318261 778213 318261 69.43 73.52 778213 356954 778213 303335 2531764 11.87 5.63 0 14.51 0 1.05 0 0.35 0 0.00 0 25.15 0 514104 0 43 0 41.94 0 1.20 0 0.01 0 1.12 0 0.01 0 167.98 0 0.37 0 39406 0 699936 0 101542 0 7378 0 2437 0 0 0 176017 0 15 0 0 0 118 0 14576 0 241 0 14950 0 58.94 0 412562 0 6096 16712 2.741469816273 699936.0 514104.0 39406.0 101542.0 7378.0 2437.0 0.0 176017.0 412562.0 73.5 5.6 14.5 1.1 0.3 0.0 25.1 58.9 43 43 43.00 38 30097248 26.3 22.8 23.1 27.9 0.0 35.9 24.4 smartseq 1065849 SRR2088437 SRP060416 SRS980064 SRX1082406 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810872: T86_P1_C10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810872 GSM1810872 T86_P1_C10_ILC3 64841936 1507952 2016-01-28 01:00:06 72618403 64841936 1507952 1 1507952 index:0,count:1507952,average:43,stdev:0 GSM1810872_r1 7.1 7.12 0.18 40533794 52131833 32553332 43107452 128.61 132.42 0 0 0 0 0 0 58.43 73.92 1530310 576357 1530310 576357 64.91 70.74 1530310 640252 1530310 551536 5788615 14.28 7.39 0 13.71 0 1.19 0 0.32 0 0.00 0 33.08 0 986387 0 43 0 41.75 0 1.15 0 0.01 0 1.14 0 0.00 0 258.51 0 0.41 0 111453 0 1507952 0 206696 0 17935 0 4843 0 0 0 498787 0 28 0 0 0 129 0 23830 0 517 0 24504 0 51.71 0 779691 0 7186 27137 3.776370720846 1507952.0 986387.0 111453.0 206696.0 17935.0 4843.0 0.0 498787.0 779691.0 65.4 7.4 13.7 1.2 0.3 0.0 33.1 51.7 43 43 43.00 38 64841936 26.3 22.4 22.9 28.4 0.0 35.2 23.5 smartseq 1065867 SRR2088438 SRP060416 SRS980063 SRX1082407 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810873: T86_P1_C11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810873 GSM1810873 T86_P1_C11_ILC3 145213408 3377056 2016-01-28 01:00:06 158361671 145213408 3377056 1 3377056 index:0,count:3377056,average:43,stdev:0 GSM1810873_r1 8.46 6.85 0.24 94218734 123952152 74520507 101172381 131.56 135.76 0 0 0 0 0 0 59.58 76.44 3631645 1365676 3631645 1365676 66.88 72.53 3631645 1532836 3631645 1295932 12077140 12.82 6.90 0 14.96 0 0.97 0 0.37 0 0.00 0 30.79 0 2292053 0 43 0 41.71 0 1.16 0 0.01 0 1.11 0 0.01 0 238.38 0 0.38 0 233084 0 3377056 0 505361 0 32872 0 12393 0 0 0 1039738 0 22 0 0 0 497 0 60130 0 1048 0 61697 0 52.91 0 1786692 0 10237 70563 6.892937383999 3377056.0 2292053.0 233084.0 505361.0 32872.0 12393.0 0.0 1039738.0 1786692.0 67.9 6.9 15.0 1.0 0.4 0.0 30.8 52.9 43 43 43.00 38 145213408 26.4 22.4 22.6 28.6 0.0 35.7 24.1 smartseq 1065884 SRR2088439 SRP060416 SRS980062 SRX1082408 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810874: T86_P1_C12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810874 GSM1810874 T86_P1_C12_ILC3 170336373 3961311 2016-01-28 01:00:06 185721189 170336373 3961311 1 3961311 index:0,count:3961311,average:43,stdev:0 GSM1810874_r1 14.68 5.37 0.17 126319172 178030406 100644528 145218576 140.94 144.29 0 0 0 0 0 0 64.91 82.17 4623420 1969472 4623420 1969472 74.8 78.9 4623420 2269701 4623420 1891029 12407689 9.82 5.02 0 16.09 0 0.75 0 0.32 0 0.00 0 22.33 0 3034179 0 43 0 41.99 0 1.14 0 0.01 0 1.12 0 0.01 0 331.64 0 0.36 0 198965 0 3961311 0 637294 0 29880 0 12527 0 0 0 884725 0 30 0 0 0 527 0 85501 0 1143 0 87201 0 60.51 0 2396885 0 15583 102265 6.562600269524 3961311.0 3034179.0 198965.0 637294.0 29880.0 12527.0 0.0 884725.0 2396885.0 76.6 5.0 16.1 0.8 0.3 0.0 22.3 60.5 43 43 43.00 38 170336373 26.4 22.6 22.8 28.1 0.0 35.7 24.5 smartseq 1065995 SRR2088440 SRP060416 SRS980061 SRX1082409 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810875: T86_P1_C1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810875 GSM1810875 T86_P1_C1_ILC3 168366586 3915502 2016-01-28 01:00:06 182687394 168366586 3915502 1 3915502 index:0,count:3915502,average:43,stdev:0 GSM1810875_r1 9.56 6.16 0.17 116099206 154695888 94465061 128783062 133.24 136.33 0 0 0 0 0 0 62.78 78.04 4188704 1758721 4188704 1758721 69.34 73.95 4188704 1942704 4188704 1666688 14037968 12.09 6.10 0 13.99 0 0.84 0 0.36 0 0.00 0 27.25 0 2801601 0 43 0 41.92 0 1.18 0 0.01 0 1.11 0 0.00 0 320.36 0 0.35 0 238929 0 3915502 0 547933 0 32719 0 14155 0 0 0 1067027 0 40 0 0 0 535 0 88009 0 1218 0 89802 0 57.56 0 2253668 0 14714 100727 6.845657197227 3915502.0 2801601.0 238929.0 547933.0 32719.0 14155.0 0.0 1067027.0 2253668.0 71.6 6.1 14.0 0.8 0.4 0.0 27.3 57.6 43 43 43.00 38 168366586 26.1 22.7 22.9 28.3 0.0 35.8 24.4 smartseq 1066012 SRR2088441 SRP060416 SRS980060 SRX1082410 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810876: T86_P1_C2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810876 GSM1810876 T86_P1_C2_ILC3 43099545 1002315 2016-01-28 01:00:06 48502846 43099545 1002315 1 1002315 index:0,count:1002315,average:43,stdev:0 GSM1810876_r1 9.81 7.36 0.21 27408610 36413072 21462694 29605339 132.85 137.94 0 0 0 0 0 0 60.17 78.08 1078891 401954 1078891 401954 67.85 74.77 1078891 453249 1078891 384926 3485774 12.72 7.17 0 15.29 0 0.83 0 0.30 0 0.00 0 32.22 0 668031 0 43 0 41.69 0 1.16 0 0.01 0 1.13 0 0.00 0 164.02 0 0.41 0 71870 0 1002315 0 153207 0 8334 0 2964 0 0 0 322986 0 6 0 0 0 120 0 16691 0 344 0 17161 0 51.36 0 514824 0 6452 19089 2.958617482951 1002315.0 668031.0 71870.0 153207.0 8334.0 2964.0 0.0 322986.0 514824.0 66.6 7.2 15.3 0.8 0.3 0.0 32.2 51.4 43 43 43.00 38 43099545 26.6 22.0 22.6 28.8 0.0 35.2 23.4 smartseq 1066027 SRR2088442 SRP060416 SRS980058 SRX1082411 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810877: T86_P1_C3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810877 GSM1810877 T86_P1_C3_ILC3 101499479 2360453 2016-01-28 01:00:06 111316539 101499479 2360453 1 2360453 index:0,count:2360453,average:43,stdev:0 GSM1810877_r1 7.84 8.68 0.23 53554033 67777210 40605485 53739244 126.56 132.34 0 0 0 0 0 0 55.66 75.14 2251403 736141 2251403 736141 62.94 72.08 2251403 832386 2251403 706136 8348232 15.59 9.56 0 14.52 0 0.98 0 0.28 0 0.00 0 42.72 0 1322459 0 43 0 41.45 0 1.16 0 0.01 0 1.11 0 0.01 0 223.62 0 0.39 0 225744 0 2360453 0 342827 0 23060 0 6577 0 0 0 1008357 0 15 0 0 0 282 0 29315 0 769 0 30381 0 41.50 0 979632 0 6218 34394 5.531360566098 2360453.0 1322459.0 225744.0 342827.0 23060.0 6577.0 0.0 1008357.0 979632.0 56.0 9.6 14.5 1.0 0.3 0.0 42.7 41.5 43 43 43.00 38 101499479 26.1 22.2 22.5 29.2 0.0 35.6 23.7 smartseq 1066045 SRR2088443 SRP060416 SRS980059 SRX1082412 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810878: T86_P1_C4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810878 GSM1810878 T86_P1_C4_ILC3 112184377 2608939 2016-01-28 01:00:06 122758888 112184377 2608939 1 2608939 index:0,count:2608939,average:43,stdev:0 GSM1810878_r1 13.34 5.93 0.22 79503791 109176119 63643416 89409339 137.32 140.48 0 0 0 0 0 0 63.73 80.5 2941699 1222298 2941699 1222298 71.45 76.91 2941699 1370338 2941699 1167747 9105259 11.45 5.69 0 15.32 0 0.76 0 0.34 0 0.00 0 25.39 0 1918020 0 43 0 41.92 0 1.12 0 0.01 0 1.11 0 0.01 0 276.24 0 0.36 0 148378 0 2608939 0 399708 0 19724 0 8774 0 0 0 662421 0 18 0 0 0 392 0 54211 0 860 0 55481 0 58.20 0 1518312 0 12001 63324 5.276560286643 2608939.0 1918020.0 148378.0 399708.0 19724.0 8774.0 0.0 662421.0 1518312.0 73.5 5.7 15.3 0.8 0.3 0.0 25.4 58.2 43 43 43.00 38 112184377 26.4 22.5 22.7 28.3 0.0 35.8 24.4 smartseq 1066063 SRR2088444 SRP060416 SRS980057 SRX1082413 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810879: T86_P1_C5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810879 GSM1810879 T86_P1_C5_ILC3 111299093 2588351 2016-01-28 01:00:06 122540549 111299093 2588351 1 2588351 index:0,count:2588351,average:43,stdev:0 GSM1810879_r1 11.22 6.57 0.19 73670329 98908639 59117405 81823734 134.26 138.41 0 0 0 0 0 0 60.02 75.86 2758160 1072483 2758160 1072483 67.58 73.2 2758160 1207460 2758160 1034865 9760141 13.25 6.68 0 14.41 0 0.87 0 0.32 0 0.00 0 29.78 0 1786792 0 43 0 41.82 0 1.11 0 0.01 0 1.13 0 0.01 0 245.21 0 0.38 0 172796 0 2588351 0 373052 0 22550 0 8234 0 0 0 770775 0 19 0 0 0 308 0 44679 0 802 0 45808 0 54.62 0 1413740 0 10070 50684 5.033167825223 2588351.0 1786792.0 172796.0 373052.0 22550.0 8234.0 0.0 770775.0 1413740.0 69.0 6.7 14.4 0.9 0.3 0.0 29.8 54.6 43 43 43.00 38 111299093 26.4 22.4 22.7 28.5 0.0 35.6 23.9 smartseq 1066078 SRR2088445 SRP060416 SRS980056 SRX1082414 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810880: T86_P1_C6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810880 GSM1810880 T86_P1_C6_ILC3 135701593 3155851 2016-01-28 01:00:06 148450779 135701593 3155851 1 3155851 index:0,count:3155851,average:43,stdev:0 GSM1810880_r1 11.32 6.75 0.2 85165720 113538914 66789795 91979107 133.32 137.71 0 0 0 0 0 0 60.75 78.69 3326905 1259178 3326905 1259178 68.78 75.3 3326905 1425649 3326905 1204994 10970982 12.88 7.45 0 14.97 0 0.84 0 0.33 0 0.00 0 33.15 0 2072856 0 43 0 41.74 0 1.11 0 0.01 0 1.14 0 0.01 0 56.81 0 0.37 0 235128 0 3155851 0 472587 0 26395 0 10363 0 0 0 1046237 0 33 0 0 0 387 0 53507 0 1021 0 54948 0 50.71 0 1600269 0 10126 62602 6.182302982421 3155851.0 2072856.0 235128.0 472587.0 26395.0 10363.0 0.0 1046237.0 1600269.0 65.7 7.5 15.0 0.8 0.3 0.0 33.2 50.7 43 43 43.00 38 135701593 26.4 22.3 22.5 28.8 0.0 35.6 24.0 smartseq 1066094 SRR2088446 SRP060416 SRS980055 SRX1082415 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810881: T86_P1_C7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810881 GSM1810881 T86_P1_C7_ILC3 148374123 3450561 2016-01-28 01:00:06 161826138 148374123 3450561 1 3450561 index:0,count:3450561,average:43,stdev:0 GSM1810881_r1 10.79 6.85 0.2 94763224 128443825 74946933 104867105 135.54 139.92 0 0 0 0 0 0 60.72 77.87 3602808 1396092 3602808 1396092 68.09 73.92 3602808 1565667 3602808 1325238 12100652 12.77 7.20 0 14.68 0 0.84 0 0.33 0 0.00 0 32.19 0 2299285 0 43 0 41.81 0 1.10 0 0.01 0 1.13 0 0.00 0 230.04 0 0.37 0 248478 0 3450561 0 506535 0 29085 0 11541 0 0 0 1110650 0 12 0 0 0 492 0 60975 0 1070 0 62549 0 51.96 0 1792750 0 10867 71749 6.602466182019 3450561.0 2299285.0 248478.0 506535.0 29085.0 11541.0 0.0 1110650.0 1792750.0 66.6 7.2 14.7 0.8 0.3 0.0 32.2 52.0 43 43 43.00 38 148374123 25.9 22.7 23.0 28.3 0.0 35.7 24.1 smartseq 1066109 SRR2088447 SRP060416 SRS980054 SRX1082416 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810882: T86_P1_C8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810882 GSM1810882 T86_P1_C8_ILC3 93841738 2182366 2016-01-28 01:00:06 103020044 93841738 2182366 1 2182366 index:0,count:2182366,average:43,stdev:0 GSM1810882_r1 11.99 5.78 0.2 64830643 87170552 52296935 72087788 134.46 137.84 0 0 0 0 0 0 61.59 77.29 2384380 965102 2384380 965102 68.71 73.7 2384380 1076656 2384380 920269 8470556 13.07 6.03 0 14.58 0 0.78 0 0.40 0 0.00 0 27.03 0 1566894 0 43 0 41.88 0 1.14 0 0.01 0 1.15 0 0.01 0 68.92 0 0.37 0 131654 0 2182366 0 318280 0 16995 0 8656 0 0 0 589821 0 24 0 0 0 302 0 43132 0 727 0 44185 0 57.21 0 1248614 0 10749 48830 4.542748162620 2182366.0 1566894.0 131654.0 318280.0 16995.0 8656.0 0.0 589821.0 1248614.0 71.8 6.0 14.6 0.8 0.4 0.0 27.0 57.2 43 43 43.00 38 93841738 26.5 22.5 22.8 28.3 0.0 35.7 24.2 smartseq 1066124 SRR2088448 SRP060416 SRS979779 SRX1082417 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810883: T86_P1_C9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810883 GSM1810883 T86_P1_C9_ILC3 29641964 689348 2016-01-28 01:00:06 32719956 29641964 689348 1 689348 index:0,count:689348,average:43,stdev:0 GSM1810883_r1 12.0 6.03 0.16 20395447 27480586 16374140 22643028 134.74 138.29 0 0 0 0 0 0 61.59 77.64 761325 303235 761325 303235 69.79 74.7 761325 343619 761325 291770 2587835 12.69 6.09 0 14.76 0 0.85 0 0.43 0 0.00 0 27.30 0 492332 0 43 0 41.92 0 1.12 0 0.01 0 1.12 0 0.00 0 137.87 0 0.36 0 41955 0 689348 0 101760 0 5853 0 2992 0 0 0 188171 0 3 0 0 0 99 0 13126 0 221 0 13449 0 56.66 0 390572 0 6395 14737 2.304456606724 689348.0 492332.0 41955.0 101760.0 5853.0 2992.0 0.0 188171.0 390572.0 71.4 6.1 14.8 0.8 0.4 0.0 27.3 56.7 43 43 43.00 38 29641964 26.5 22.5 22.7 28.3 0.0 35.8 24.4 smartseq 1066141 SRR2088449 SRP060416 SRS980053 SRX1082418 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810884: T86_P1_D10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810884 GSM1810884 T86_P1_D10_ILC3 28320617 658619 2016-01-28 01:00:06 31270324 28320617 658619 1 658619 index:0,count:658619,average:43,stdev:0 GSM1810884_r1 7.17 6.91 0.23 17163606 22176436 13708630 18340336 129.21 133.79 0 0 0 0 0 0 56.06 71.33 659688 234752 659688 234752 62.47 68.42 659688 261582 659688 225176 2598860 15.14 7.71 0 13.61 0 1.35 0 0.39 0 0.00 0 34.68 0 418758 0 43 0 41.66 0 1.16 0 0.01 0 1.12 0 0.00 0 169.36 0 0.38 0 50791 0 658619 0 89663 0 8885 0 2554 0 0 0 228422 0 3 0 0 0 83 0 9794 0 214 0 10094 0 49.97 0 329095 0 4359 11240 2.578573067217 658619.0 418758.0 50791.0 89663.0 8885.0 2554.0 0.0 228422.0 329095.0 63.6 7.7 13.6 1.3 0.4 0.0 34.7 50.0 43 43 43.00 38 28320617 26.3 22.5 22.7 28.4 0.0 35.7 24.0 smartseq 1066252 SRR2088450 SRP060416 SRS980051 SRX1082419 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810885: T86_P1_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810885 GSM1810885 T86_P1_D11_NK 57716363 1342241 2016-01-28 01:00:06 63195761 57716363 1342241 1 1342241 index:0,count:1342241,average:43,stdev:0 GSM1810885_r1 6.59 6.81 0.22 35034670 44441093 27032569 35463528 126.85 131.19 0 0 0 0 0 0 54.89 72.33 1426236 469779 1426236 469779 62.87 68.63 1426236 538085 1426236 445792 5765616 16.46 7.75 0 15.37 0 1.06 0 0.36 0 0.00 0 34.82 0 855846 0 43 0 41.62 0 1.20 0 0.01 0 1.13 0 0.00 0 219.64 0 0.37 0 104022 0 1342241 0 206308 0 14203 0 4857 0 0 0 467335 0 12 0 0 0 156 0 20613 0 415 0 21196 0 48.39 0 649538 0 6370 24664 3.871899529042 1342241.0 855846.0 104022.0 206308.0 14203.0 4857.0 0.0 467335.0 649538.0 63.8 7.7 15.4 1.1 0.4 0.0 34.8 48.4 43 43 43.00 38 57716363 26.4 22.4 22.5 28.7 0.0 35.8 24.2 smartseq 1066268 SRR2088451 SRP060416 SRS980052 SRX1082420 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810886: T86_P1_D12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810886 GSM1810886 T86_P1_D12_ILC3 69844470 1624290 2016-01-28 01:00:06 76276378 69844470 1624290 1 1624290 index:0,count:1624290,average:43,stdev:0 GSM1810886_r1 9.59 6.92 0.22 38517788 51039462 30227709 41318786 132.51 136.69 0 0 0 0 0 0 59.76 77.29 1494609 560280 1494609 560280 66.9 73.06 1494609 627221 1494609 529626 5313609 13.80 9.39 0 13.09 0 0.72 0 0.24 0 0.00 0 41.33 0 937529 0 43 0 41.70 0 1.19 0 0.01 0 1.10 0 0.00 0 188.63 0 0.36 0 152457 0 1624290 0 212644 0 11624 0 3882 0 0 0 671255 0 23 0 0 0 254 0 26623 0 566 0 27466 0 44.63 0 724885 0 8305 29814 3.589885611078 1624290.0 937529.0 152457.0 212644.0 11624.0 3882.0 0.0 671255.0 724885.0 57.7 9.4 13.1 0.7 0.2 0.0 41.3 44.6 43 43 43.00 38 69844470 26.9 22.1 22.4 28.7 0.0 35.9 24.5 smartseq 1066284 SRR2088452 SRP060416 SRS980050 SRX1082421 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810887: T86_P1_D1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810887 GSM1810887 T86_P1_D1_ILC3 55480363 1290241 2016-01-28 01:00:06 60843594 55480363 1290241 1 1290241 index:0,count:1290241,average:43,stdev:0 GSM1810887_r1 8.3 7.31 0.22 32574716 42168836 25253644 34093074 129.45 135.0 0 0 0 0 0 0 56.94 74.79 1307567 453848 1307567 453848 64.02 71.55 1307567 510299 1307567 434177 4532938 13.92 8.11 0 14.74 0 1.53 0 0.27 0 0.00 0 36.42 0 797037 0 43 0 41.62 0 1.11 0 0.01 0 1.10 0 0.00 0 232.24 0 0.39 0 104703 0 1290241 0 190205 0 19791 0 3509 0 0 0 469904 0 10 0 0 0 134 0 17838 0 418 0 18400 0 47.03 0 606832 0 5853 20465 3.496497522638 1290241.0 797037.0 104703.0 190205.0 19791.0 3509.0 0.0 469904.0 606832.0 61.8 8.1 14.7 1.5 0.3 0.0 36.4 47.0 43 43 43.00 38 55480363 26.3 22.4 22.6 28.8 0.0 35.8 24.1 smartseq 1066299 SRR2088453 SRP060416 SRS980049 SRX1082422 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810888: T86_P1_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810888 GSM1810888 T86_P1_D2_NK 42454674 987318 2016-01-28 01:00:06 46653364 42454674 987318 1 987318 index:0,count:987318,average:43,stdev:0 GSM1810888_r1 8.88 6.28 0.17 28391044 38041798 22760146 31454893 133.99 138.2 0 0 0 0 0 0 64.84 81.94 1079784 446204 1079784 446204 71.76 78.05 1079784 493788 1079784 425006 3047007 10.73 6.50 0 14.54 0 0.79 0 0.30 0 0.00 0 29.21 0 688145 0 43 0 41.80 0 1.18 0 0.01 0 1.11 0 0.01 0 161.56 0 0.37 0 64211 0 987318 0 143603 0 7830 0 2986 0 0 0 288357 0 13 0 0 0 193 0 22022 0 280 0 22508 0 55.15 0 544542 0 8231 24476 2.973636253189 987318.0 688145.0 64211.0 143603.0 7830.0 2986.0 0.0 288357.0 544542.0 69.7 6.5 14.5 0.8 0.3 0.0 29.2 55.2 43 43 43.00 38 42454674 26.1 22.8 22.9 28.2 0.0 35.9 24.3 smartseq 1066314 SRR2088454 SRP060416 SRS980048 SRX1082423 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810889: T86_P1_D3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810889 GSM1810889 T86_P1_D3_ILC3 82763820 1924740 2016-01-28 01:00:06 90045923 82763820 1924740 1 1924740 index:0,count:1924740,average:43,stdev:0 GSM1810889_r1 13.19 4.42 0.18 65663178 92885429 51694796 74897182 141.46 144.88 0 0 0 0 0 0 65.01 83.09 2457237 1022226 2457237 1022226 75.52 79.16 2457237 1187391 2457237 973896 5770935 8.79 3.78 0 17.77 0 1.05 0 0.42 0 0.00 0 16.84 0 1572339 0 43 0 42.02 0 1.11 0 0.01 0 1.12 0 0.00 0 301.26 0 0.36 0 72715 0 1924740 0 342062 0 20194 0 8036 0 0 0 324171 0 11 0 0 0 317 0 44701 0 573 0 45602 0 63.92 0 1230277 0 12921 53557 4.144957820602 1924740.0 1572339.0 72715.0 342062.0 20194.0 8036.0 0.0 324171.0 1230277.0 81.7 3.8 17.8 1.0 0.4 0.0 16.8 63.9 43 43 43.00 38 82763820 26.2 23.1 23.2 27.5 0.0 36.1 25.1 smartseq 1066330 SRR2088455 SRP060416 SRS980047 SRX1082424 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810890: T86_P1_D5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810890 GSM1810890 T86_P1_D5_ILC3 52571542 1222594 2016-01-28 01:00:06 57638043 52571542 1222594 1 1222594 index:0,count:1222594,average:43,stdev:0 GSM1810890_r1 6.54 6.71 0.23 34063987 43250054 27111878 35384410 126.97 130.51 0 0 0 0 0 0 57.17 72.8 1310217 472382 1310217 472382 63.98 69.1 1310217 528673 1310217 448379 5034951 14.78 6.95 0 14.51 0 0.96 0 0.47 0 0.00 0 30.99 0 826250 0 43 0 41.78 0 1.18 0 0.01 0 1.13 0 0.00 0 244.52 0 0.38 0 84951 0 1222594 0 177374 0 11730 0 5771 0 0 0 378843 0 9 0 0 0 185 0 22722 0 468 0 23384 0 53.07 0 648876 0 8255 26368 3.194185342217 1222594.0 826250.0 84951.0 177374.0 11730.0 5771.0 0.0 378843.0 648876.0 67.6 6.9 14.5 1.0 0.5 0.0 31.0 53.1 43 43 43.00 38 52571542 26.4 22.5 22.7 28.4 0.0 35.9 24.4 smartseq 1066346 SRR2088456 SRP060416 SRS980046 SRX1082425 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810891: T86_P1_D6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810891 GSM1810891 T86_P1_D6_ILC3 53120867 1235369 2016-01-28 01:00:06 58120835 53120867 1235369 1 1235369 index:0,count:1235369,average:43,stdev:0 GSM1810891_r1 6.84 5.91 0.17 36206906 46919681 29180994 38834427 129.59 133.08 0 0 0 0 0 0 61.56 77.35 1355044 540081 1355044 540081 68.17 73.38 1355044 598122 1355044 512352 4616570 12.75 6.18 0 14.50 0 0.87 0 0.35 0 0.00 0 27.75 0 877354 0 43 0 41.79 0 1.14 0 0.01 0 1.11 0 0.00 0 261.61 0 0.36 0 76306 0 1235369 0 179149 0 10777 0 4380 0 0 0 342858 0 15 0 0 0 199 0 27233 0 387 0 27834 0 56.52 0 698205 0 9755 31535 3.232701178883 1235369.0 877354.0 76306.0 179149.0 10777.0 4380.0 0.0 342858.0 698205.0 71.0 6.2 14.5 0.9 0.4 0.0 27.8 56.5 43 43 43.00 38 53120867 26.3 22.6 22.7 28.3 0.0 35.9 24.4 smartseq 1066362 SRR2088457 SRP060416 SRS980045 SRX1082426 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810892: T86_P1_D7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810892 GSM1810892 T86_P1_D7_ILC3 83712658 1946806 2016-01-28 01:00:06 91391660 83712658 1946806 1 1946806 index:0,count:1946806,average:43,stdev:0 GSM1810892_r1 14.01 5.32 0.15 61090933 85891825 49698982 71430582 140.6 143.73 0 0 0 0 0 0 65.56 81.33 2174504 963908 2174504 963908 73.48 77.53 2174504 1080378 2174504 918880 6610276 10.82 5.23 0 14.64 0 0.75 0 0.34 0 0.00 0 23.38 0 1470275 0 43 0 41.93 0 1.12 0 0.01 0 1.11 0 0.00 0 259.57 0 0.36 0 101911 0 1946806 0 285099 0 14644 0 6711 0 0 0 455176 0 20 0 0 0 333 0 41303 0 655 0 42311 0 60.88 0 1185176 0 11392 47498 4.169417134831 1946806.0 1470275.0 101911.0 285099.0 14644.0 6711.0 0.0 455176.0 1185176.0 75.5 5.2 14.6 0.8 0.3 0.0 23.4 60.9 43 43 43.00 38 83712658 26.2 22.9 23.1 27.8 0.0 35.9 24.6 smartseq 1066378 SRR2088458 SRP060416 SRS980042 SRX1082427 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810893: T86_P1_D8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810893 GSM1810893 T86_P1_D8_ILC3 40275735 936645 2016-01-28 01:00:06 44396115 40275735 936645 1 936645 index:0,count:936645,average:43,stdev:0 GSM1810893_r1 6.54 6.78 0.18 25551291 33174730 20617185 27729566 129.84 134.5 0 0 0 0 0 0 59.24 74.5 973671 367873 973671 367873 64.69 70.66 973671 401697 973671 348897 3519779 13.78 7.10 0 13.58 0 1.45 0 0.35 0 0.00 0 31.91 0 621002 0 43 0 41.75 0 1.18 0 0.01 0 1.12 0 0.00 0 187.33 0 0.38 0 66482 0 936645 0 127234 0 13549 0 3236 0 0 0 298858 0 4 0 0 0 140 0 17257 0 325 0 17726 0 52.72 0 493768 0 6560 18982 2.893597560976 936645.0 621002.0 66482.0 127234.0 13549.0 3236.0 0.0 298858.0 493768.0 66.3 7.1 13.6 1.4 0.3 0.0 31.9 52.7 43 43 43.00 38 40275735 26.2 22.7 22.9 28.1 0.0 35.7 24.1 smartseq 1066394 SRR2088459 SRP060416 SRS980043 SRX1082428 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810894: T86_P1_E10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810894 GSM1810894 T86_P1_E10_ILC3 55959641 1301387 2016-01-28 01:00:06 61631816 55959641 1301387 1 1301387 index:0,count:1301387,average:43,stdev:0 GSM1810894_r1 8.38 6.21 0.19 37309373 49156529 30367474 41120466 131.75 135.41 0 0 0 0 0 0 62.05 77.21 1368998 561094 1368998 561094 68.58 73.8 1368998 620135 1368998 536309 4763027 12.77 6.58 0 13.64 0 0.87 0 0.36 0 0.00 0 29.29 0 904250 0 43 0 41.79 0 1.17 0 0.01 0 1.11 0 0.01 0 292.81 0 0.38 0 85635 0 1301387 0 177571 0 11262 0 4643 0 0 0 381232 0 6 0 0 0 165 0 24159 0 430 0 24760 0 55.84 0 726679 0 7738 27321 3.530757301628 1301387.0 904250.0 85635.0 177571.0 11262.0 4643.0 0.0 381232.0 726679.0 69.5 6.6 13.6 0.9 0.4 0.0 29.3 55.8 43 43 43.00 38 55959641 26.3 22.6 22.9 28.2 0.0 35.7 24.1 smartseq 1066505 SRR2088460 SRP060416 SRS979876 SRX1082429 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810895: T86_P1_E11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810895 GSM1810895 T86_P1_E11_ILC3 124383864 2892648 2016-01-28 01:00:06 135332887 124383864 2892648 1 2892648 index:0,count:2892648,average:43,stdev:0 GSM1810895_r1 13.41 6.49 0.2 81542097 110005422 65603489 90711920 134.91 138.27 0 0 0 0 0 0 60.86 76.68 3007185 1204113 3007185 1204113 68.7 73.84 3007185 1359081 3007185 1159619 11075375 13.58 6.78 0 14.11 0 0.83 0 0.36 0 0.00 0 30.42 0 1978424 0 43 0 41.77 0 1.11 0 0.01 0 1.12 0 0.00 0 289.26 0 0.37 0 196006 0 2892648 0 408015 0 23875 0 10426 0 0 0 879923 0 15 0 0 0 350 0 47554 0 835 0 48754 0 54.29 0 1570409 0 10058 54487 5.417279777292 2892648.0 1978424.0 196006.0 408015.0 23875.0 10426.0 0.0 879923.0 1570409.0 68.4 6.8 14.1 0.8 0.4 0.0 30.4 54.3 43 43 43.00 38 124383864 26.5 22.4 22.5 28.5 0.0 35.8 24.3 smartseq 1066521 SRR2088461 SRP060416 SRS979875 SRX1082430 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810896: T86_P1_E12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810896 GSM1810896 T86_P1_E12_ILC3 170657712 3968784 2016-01-28 01:00:06 184448814 170657712 3968784 1 3968784 index:0,count:3968784,average:43,stdev:0 GSM1810896_r1 12.46 5.75 0.16 122771846 168728478 99156457 139552606 137.43 140.74 0 0 0 0 0 0 64.53 80.66 4448122 1908788 4448122 1908788 72.61 77.03 4448122 2147980 4448122 1822852 12645691 10.30 5.40 0 14.91 0 0.85 0 0.40 0 0.00 0 24.22 0 2958122 0 43 0 41.90 0 1.13 0 0.01 0 1.14 0 0.00 0 91.59 0 0.37 0 214504 0 3968784 0 591722 0 33574 0 15730 0 0 0 961358 0 44 0 0 0 604 0 88150 0 1257 0 90055 0 59.63 0 2366400 0 14693 102605 6.983257333424 3968784.0 2958122.0 214504.0 591722.0 33574.0 15730.0 0.0 961358.0 2366400.0 74.5 5.4 14.9 0.8 0.4 0.0 24.2 59.6 43 43 43.00 38 170657712 26.2 22.9 23.1 27.8 0.0 35.9 24.5 smartseq 1066538 SRR2088462 SRP060416 SRS979874 SRX1082431 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810897: T86_P1_E1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810897 GSM1810897 T86_P1_E1_ILC3 109672446 2550522 2016-01-28 01:00:06 119700076 109672446 2550522 1 2550522 index:0,count:2550522,average:43,stdev:0 GSM1810897_r1 5.96 7.1 0.22 68477250 87191608 54115535 70949049 127.33 131.11 0 0 0 0 0 0 58.9 75.69 2653751 982468 2653751 982468 66.14 72.08 2653751 1103358 2653751 935631 9089187 13.27 7.48 0 14.51 0 0.91 0 0.26 0 0.00 0 33.43 0 1668103 0 43 0 41.69 0 1.19 0 0.01 0 1.11 0 0.00 0 229.55 0 0.38 0 190767 0 2550522 0 370098 0 23281 0 6625 0 0 0 852513 0 20 0 0 0 426 0 44876 0 820 0 46142 0 50.89 0 1298005 0 8607 52465 6.095619844313 2550522.0 1668103.0 190767.0 370098.0 23281.0 6625.0 0.0 852513.0 1298005.0 65.4 7.5 14.5 0.9 0.3 0.0 33.4 50.9 43 43 43.00 38 109672446 26.2 22.6 22.7 28.5 0.0 35.7 24.0 smartseq 1066553 SRR2088463 SRP060416 SRS979873 SRX1082432 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810898: T86_P1_E2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810898 GSM1810898 T86_P1_E2_ILC3 48893580 1137060 2016-01-28 01:00:06 54533445 48893580 1137060 1 1137060 index:0,count:1137060,average:43,stdev:0 GSM1810898_r1 6.37 7.64 0.23 28105354 34704962 21848529 27989326 123.48 128.11 0 0 0 0 0 0 54.57 71.64 1141576 376739 1141576 376739 61.17 68.49 1141576 422275 1141576 360176 4980900 17.72 8.44 0 14.46 0 0.96 0 0.35 0 0.00 0 37.97 0 690329 0 43 0 41.55 0 1.17 0 0.01 0 1.12 0 0.01 0 272.89 0 0.40 0 95935 0 1137060 0 164472 0 10950 0 3989 0 0 0 431792 0 8 0 0 0 146 0 16227 0 353 0 16734 0 46.25 0 525857 0 5610 18479 3.293939393939 1137060.0 690329.0 95935.0 164472.0 10950.0 3989.0 0.0 431792.0 525857.0 60.7 8.4 14.5 1.0 0.4 0.0 38.0 46.2 43 43 43.00 38 48893580 26.5 22.0 22.3 29.1 0.0 35.3 23.4 smartseq 1066569 SRR2088464 SRP060416 SRS979872 SRX1082433 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810899: T86_P1_E3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810899 GSM1810899 T86_P1_E3_ILC3 122319864 2844648 2016-01-28 01:00:06 133139348 122319864 2844648 1 2844648 index:0,count:2844648,average:43,stdev:0 GSM1810899_r1 9.37 6.64 0.17 76066119 99756187 61150116 82565967 131.14 135.02 0 0 0 0 0 0 62.17 78.48 2860864 1150572 2860864 1150572 68.33 74.64 2860864 1264534 2860864 1094332 9520316 12.52 7.58 0 13.52 0 0.89 0 0.34 0 0.00 0 33.71 0 1850639 0 43 0 41.71 0 1.16 0 0.01 0 1.15 0 0.00 0 249.77 0 0.38 0 215758 0 2844648 0 384509 0 25415 0 9548 0 0 0 959046 0 21 0 0 0 455 0 57610 0 951 0 59037 0 51.54 0 1466130 0 12453 65398 5.251585963222 2844648.0 1850639.0 215758.0 384509.0 25415.0 9548.0 0.0 959046.0 1466130.0 65.1 7.6 13.5 0.9 0.3 0.0 33.7 51.5 43 43 43.00 38 122319864 26.3 22.6 22.8 28.3 0.0 35.8 24.2 smartseq 1066585 SRR2088465 SRP060416 SRS979871 SRX1082434 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810900: T86_P1_E4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810900 GSM1810900 T86_P1_E4_ILC3 95540797 2221879 2016-01-28 01:00:06 106118668 95540797 2221879 1 2221879 index:0,count:2221879,average:43,stdev:0 GSM1810900_r1 9.53 6.19 0.17 66585252 89773786 53911386 74709420 134.83 138.58 0 0 0 0 0 0 63.25 79.04 2437681 1016327 2437681 1016327 70.39 75.53 2437681 1130948 2437681 971192 7789432 11.70 5.92 0 14.44 0 0.84 0 0.33 0 0.00 0 26.51 0 1606719 0 43 0 41.92 0 1.13 0 0.01 0 1.14 0 0.01 0 222.19 0 0.40 0 131512 0 2221879 0 320809 0 18745 0 7443 0 0 0 588972 0 33 0 0 0 367 0 45910 0 642 0 46952 0 57.87 0 1285910 0 10884 52457 4.819643513414 2221879.0 1606719.0 131512.0 320809.0 18745.0 7443.0 0.0 588972.0 1285910.0 72.3 5.9 14.4 0.8 0.3 0.0 26.5 57.9 43 43 43.00 38 95540797 26.4 22.5 23.0 28.1 0.0 35.4 23.8 smartseq 1066601 SRR2088466 SRP060416 SRS979870 SRX1082435 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810901: T86_P1_E5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810901 GSM1810901 T86_P1_E5_ILC3 62843812 1461484 2016-01-28 01:00:06 69045465 62843812 1461484 1 1461484 index:0,count:1461484,average:43,stdev:0 GSM1810901_r1 8.97 7.42 0.2 40866231 53972943 32739239 44779630 132.07 136.78 0 0 0 0 0 0 59.69 75.68 1542897 594183 1542897 594183 65.91 72.13 1542897 656108 1542897 566279 5539705 13.56 6.89 0 14.39 0 0.83 0 0.35 0 0.00 0 30.70 0 995447 0 43 0 41.70 0 1.16 0 0.01 0 1.12 0 0.01 0 276.91 0 0.38 0 100660 0 1461484 0 210352 0 12168 0 5175 0 0 0 448694 0 8 0 0 0 187 0 26614 0 511 0 27320 0 53.72 0 785095 0 8257 30585 3.704129829236 1461484.0 995447.0 100660.0 210352.0 12168.0 5175.0 0.0 448694.0 785095.0 68.1 6.9 14.4 0.8 0.4 0.0 30.7 53.7 43 43 43.00 38 62843812 26.5 22.3 22.4 28.8 0.0 35.7 24.0 smartseq 1066617 SRR2088467 SRP060416 SRS979869 SRX1082436 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810902: T86_P1_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810902 GSM1810902 T86_P1_E6_NK 120402150 2800050 2016-01-28 01:00:06 131172683 120402150 2800050 1 2800050 index:0,count:2800050,average:43,stdev:0 GSM1810902_r1 7.32 7.18 0.21 73473524 95799293 57642334 78268356 130.39 135.78 0 0 0 0 0 0 60.01 77.8 2926094 1077400 2926094 1077400 66.05 73.44 2926094 1185842 2926094 1016984 10306099 14.03 7.73 0 14.67 0 0.99 0 0.33 0 0.00 0 34.55 0 1795481 0 43 0 41.63 0 1.14 0 0.01 0 1.12 0 0.00 0 272.44 0 0.38 0 216376 0 2800050 0 410699 0 27821 0 9294 0 0 0 967454 0 38 0 0 0 374 0 52346 0 905 0 53663 0 49.46 0 1384782 0 9023 60342 6.687576194170 2800050.0 1795481.0 216376.0 410699.0 27821.0 9294.0 0.0 967454.0 1384782.0 64.1 7.7 14.7 1.0 0.3 0.0 34.6 49.5 43 43 43.00 38 120402150 26.1 22.5 22.7 28.8 0.0 35.7 23.9 smartseq 1066633 SRR2088468 SRP060416 SRS979868 SRX1082437 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810903: T86_P1_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810903 GSM1810903 T86_P1_E7_NK 112225614 2609898 2016-01-28 01:00:06 122574829 112225614 2609898 1 2609898 index:0,count:2609898,average:43,stdev:0 GSM1810903_r1 7.6 6.28 0.21 73772468 97125594 58193780 79209780 131.66 136.11 0 0 0 0 0 0 62.24 79.94 2880651 1114048 2880651 1114048 69.58 75.68 2880651 1245499 2880651 1054614 8457996 11.46 6.77 0 15.18 0 0.90 0 0.29 0 0.00 0 30.22 0 1789904 0 43 0 41.76 0 1.18 0 0.01 0 1.17 0 0.01 0 335.56 0 0.38 0 176691 0 2609898 0 396308 0 23566 0 7596 0 0 0 788832 0 24 0 0 0 301 0 53384 0 821 0 54530 0 53.40 0 1393596 0 9943 61594 6.194709846123 2609898.0 1789904.0 176691.0 396308.0 23566.0 7596.0 0.0 788832.0 1393596.0 68.6 6.8 15.2 0.9 0.3 0.0 30.2 53.4 43 43 43.00 38 112225614 26.2 22.7 22.9 28.2 0.0 35.7 24.1 smartseq 1066650 SRR2088469 SRP060416 SRS979867 SRX1082438 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810904: T86_P1_E8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810904 GSM1810904 T86_P1_E8_ILC3 73661408 1713056 2016-01-28 01:00:06 80938998 73661408 1713056 1 1713056 index:0,count:1713056,average:43,stdev:0 GSM1810904_r1 11.25 6.02 0.21 50836233 68546539 41095152 56799895 134.84 138.22 0 0 0 0 0 0 62.25 77.88 1861799 764850 1861799 764850 69.93 74.82 1861799 859177 1861799 734801 6078860 11.96 5.98 0 14.40 0 1.10 0 0.35 0 0.00 0 26.82 0 1228710 0 43 0 41.84 0 1.12 0 0.01 0 1.13 0 0.01 0 205.57 0 0.38 0 102398 0 1713056 0 246601 0 18916 0 6071 0 0 0 459359 0 16 0 0 0 261 0 32847 0 545 0 33669 0 57.33 0 982109 0 9143 37829 4.137482226840 1713056.0 1228710.0 102398.0 246601.0 18916.0 6071.0 0.0 459359.0 982109.0 71.7 6.0 14.4 1.1 0.4 0.0 26.8 57.3 43 43 43.00 38 73661408 26.3 22.7 22.9 28.1 0.0 35.7 24.2 smartseq 1066763 SRR2088470 SRP060416 SRS979866 SRX1082439 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810905: T86_P1_E9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810905 GSM1810905 T86_P1_E9_ILC3 24901386 579102 2016-01-28 01:00:06 27454711 24901386 579102 1 579102 index:0,count:579102,average:43,stdev:0 GSM1810905_r1 8.66 6.07 0.18 17400368 22882937 14419247 19421032 131.51 134.69 0 0 0 0 0 0 59.63 72.79 616044 250265 616044 250265 64.98 69.16 616044 272724 616044 237792 2665064 15.32 5.90 0 13.10 0 0.87 0 0.49 0 0.00 0 26.17 0 419680 0 43 0 41.94 0 1.17 0 0.01 0 1.16 0 0.01 0 122.63 0 0.36 0 34194 0 579102 0 75849 0 5048 0 2828 0 0 0 151546 0 5 0 0 0 91 0 11176 0 191 0 11463 0 59.37 0 343831 0 5959 12346 2.071824131566 579102.0 419680.0 34194.0 75849.0 5048.0 2828.0 0.0 151546.0 343831.0 72.5 5.9 13.1 0.9 0.5 0.0 26.2 59.4 43 43 43.00 38 24901386 26.6 22.5 22.7 28.2 0.0 35.9 24.4 smartseq 1066777 SRR2088471 SRP060416 SRS979865 SRX1082440 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810906: T86_P1_F10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810906 GSM1810906 T86_P1_F10_ILC3 69421823 1614461 2016-01-28 01:00:06 76254592 69421823 1614461 1 1614461 index:0,count:1614461,average:43,stdev:0 GSM1810906_r1 11.54 5.04 0.21 50830879 70225911 41185036 58310241 138.16 141.58 0 0 0 0 0 0 66.63 82.98 1826951 815712 1826951 815712 74.27 78.72 1826951 909305 1826951 773815 4969233 9.78 5.18 0 14.94 0 0.76 0 0.28 0 0.00 0 23.13 0 1224293 0 43 0 41.90 0 1.13 0 0.01 0 1.14 0 0.00 0 61.83 0 0.37 0 83582 0 1614461 0 241245 0 12297 0 4488 0 0 0 373383 0 26 0 0 0 242 0 39225 0 477 0 39970 0 60.89 0 983048 0 11698 46160 3.945973670713 1614461.0 1224293.0 83582.0 241245.0 12297.0 4488.0 0.0 373383.0 983048.0 75.8 5.2 14.9 0.8 0.3 0.0 23.1 60.9 43 43 43.00 38 69421823 26.4 22.7 22.9 27.9 0.0 35.8 24.5 smartseq 1066794 SRR2088472 SRP060416 SRS979864 SRX1082441 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810907: T86_P1_F11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810907 GSM1810907 T86_P1_F11_ILC3 103410270 2404890 2016-01-28 01:00:06 112923776 103410270 2404890 1 2404890 index:0,count:2404890,average:43,stdev:0 GSM1810907_r1 9.44 7.17 0.23 65643384 86103487 52852288 71601795 131.17 135.48 0 0 0 0 0 0 60.75 76.62 2464595 971175 2464595 971175 66.88 73.04 2464595 1069083 2464595 925783 9284016 14.14 7.23 0 13.76 0 0.93 0 0.35 0 0.00 0 32.25 0 1598582 0 43 0 41.70 0 1.13 0 0.01 0 1.12 0 0.01 0 320.65 0 0.37 0 173837 0 2404890 0 331028 0 22462 0 8377 0 0 0 775469 0 4 0 0 0 329 0 43551 0 788 0 44672 0 52.71 0 1267554 0 9527 49709 5.217697071481 2404890.0 1598582.0 173837.0 331028.0 22462.0 8377.0 0.0 775469.0 1267554.0 66.5 7.2 13.8 0.9 0.3 0.0 32.2 52.7 43 43 43.00 38 103410270 26.4 22.4 22.5 28.7 0.0 35.7 24.1 smartseq 1066809 SRR2088473 SRP060416 SRS979780 SRX1082442 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810908: T86_P1_F12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810908 GSM1810908 T86_P1_F12_ILC3 127896491 2974337 2016-01-28 01:00:06 139028505 127896491 2974337 1 2974337 index:0,count:2974337,average:43,stdev:0 GSM1810908_r1 10.42 6.15 0.22 87954610 117566775 70097553 96540463 133.67 137.72 0 0 0 0 0 0 62.57 79.48 3350039 1333108 3350039 1333108 70.31 75.78 3350039 1497919 3350039 1271190 10187359 11.58 6.05 0 15.24 0 0.87 0 0.32 0 0.00 0 27.18 0 2130559 0 43 0 41.79 0 1.11 0 0.01 0 1.12 0 0.00 0 254.94 0 0.37 0 180020 0 2974337 0 453187 0 25773 0 9646 0 0 0 808359 0 47 0 0 0 420 0 59394 0 975 0 60836 0 56.39 0 1677372 0 11070 68373 6.176422764228 2974337.0 2130559.0 180020.0 453187.0 25773.0 9646.0 0.0 808359.0 1677372.0 71.6 6.1 15.2 0.9 0.3 0.0 27.2 56.4 43 43 43.00 38 127896491 26.4 22.5 22.7 28.4 0.0 35.8 24.3 smartseq 1066825 SRR2088474 SRP060416 SRS979863 SRX1082443 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810909: T86_P1_F1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810909 GSM1810909 T86_P1_F1_ILC3 112319870 2612090 2016-01-28 01:00:06 122415724 112319870 2612090 1 2612090 index:0,count:2612090,average:43,stdev:0 GSM1810909_r1 8.56 6.87 0.2 72227924 94714662 57361590 77490256 131.13 135.09 0 0 0 0 0 0 60.59 77.41 2752133 1063869 2752133 1063869 67.84 73.61 2752133 1191110 2752133 1011629 9103111 12.60 7.08 0 14.60 0 0.83 0 0.30 0 0.00 0 31.66 0 1755715 0 43 0 41.74 0 1.15 0 0.01 0 1.12 0 0.01 0 261.21 0 0.37 0 184967 0 2612090 0 381455 0 21725 0 7744 0 0 0 826906 0 11 0 0 0 335 0 49092 0 814 0 50252 0 52.61 0 1374260 0 9093 57127 6.282525019246 2612090.0 1755715.0 184967.0 381455.0 21725.0 7744.0 0.0 826906.0 1374260.0 67.2 7.1 14.6 0.8 0.3 0.0 31.7 52.6 43 43 43.00 38 112319870 26.3 22.5 22.6 28.6 0.0 35.8 24.1 smartseq 1066841 SRR2088475 SRP060416 SRS979862 SRX1082444 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810910: T86_P1_F2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810910 GSM1810910 T86_P1_F2_ILC3 56745122 1319654 2016-01-28 01:00:06 63241192 56745122 1319654 1 1319654 index:0,count:1319654,average:43,stdev:0 GSM1810910_r1 9.19 6.32 0.17 37785024 49418933 30584481 41181641 130.79 134.65 0 0 0 0 0 0 59.97 75.11 1414277 549297 1414277 549297 66.28 71.8 1414277 607096 1414277 525088 5125889 13.57 6.57 0 13.99 0 0.82 0 0.36 0 0.00 0 29.41 0 915956 0 43 0 41.82 0 1.15 0 0.01 0 1.16 0 0.01 0 296.92 0 0.39 0 86751 0 1319654 0 184643 0 10845 0 4710 0 0 0 388143 0 5 0 0 0 185 0 23830 0 397 0 24417 0 55.42 0 731313 0 8086 26238 3.244867672520 1319654.0 915956.0 86751.0 184643.0 10845.0 4710.0 0.0 388143.0 731313.0 69.4 6.6 14.0 0.8 0.4 0.0 29.4 55.4 43 43 43.00 38 56745122 26.5 22.3 22.7 28.5 0.0 35.4 23.7 smartseq 1066857 SRR2088476 SRP060416 SRS979861 SRX1082445 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810911: T86_P1_F3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810911 GSM1810911 T86_P1_F3_ILC3 122903073 2858211 2016-01-28 01:00:06 133645923 122903073 2858211 1 2858211 index:0,count:2858211,average:43,stdev:0 GSM1810911_r1 12.62 5.67 0.16 92232008 126887187 74735250 104941432 137.57 140.42 0 0 0 0 0 0 64.87 80.73 3316875 1437439 3316875 1437439 72.8 76.64 3316875 1613272 3316875 1364527 9237154 10.02 4.79 0 15.24 0 0.77 0 0.34 0 0.00 0 21.36 0 2216040 0 43 0 41.97 0 1.14 0 0.01 0 1.14 0 0.00 0 342.99 0 0.35 0 136879 0 2858211 0 435558 0 21917 0 9776 0 0 0 610478 0 33 0 0 0 517 0 64787 0 823 0 66160 0 62.29 0 1780482 0 15219 75975 4.992115119259 2858211.0 2216040.0 136879.0 435558.0 21917.0 9776.0 0.0 610478.0 1780482.0 77.5 4.8 15.2 0.8 0.3 0.0 21.4 62.3 43 43 43.00 38 122903073 26.4 22.7 22.9 28.0 0.0 35.9 24.7 smartseq 1066873 SRR2088477 SRP060416 SRS979858 SRX1082446 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810912: T86_P1_F4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810912 GSM1810912 T86_P1_F4_ILC3 95919713 2230691 2016-01-28 01:00:06 104751418 95919713 2230691 1 2230691 index:0,count:2230691,average:43,stdev:0 GSM1810912_r1 12.75 5.71 0.22 65897053 89443522 52527675 73113022 135.73 139.19 0 0 0 0 0 0 60.5 76.79 2477963 962886 2477963 962886 68.33 72.88 2477963 1087575 2477963 913850 8512536 12.92 6.20 0 15.14 0 0.80 0 0.33 0 0.00 0 27.52 0 1591542 0 43 0 41.89 0 1.10 0 0.01 0 1.15 0 0.01 0 223.07 0 0.37 0 138239 0 2230691 0 337676 0 17820 0 7440 0 0 0 613889 0 5 0 0 0 305 0 40938 0 681 0 41929 0 56.21 0 1253866 0 9880 47266 4.784008097166 2230691.0 1591542.0 138239.0 337676.0 17820.0 7440.0 0.0 613889.0 1253866.0 71.3 6.2 15.1 0.8 0.3 0.0 27.5 56.2 43 43 43.00 38 95919713 26.5 22.5 22.6 28.5 0.0 35.8 24.4 smartseq 1066889 SRR2088478 SRP060416 SRS979860 SRX1082447 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810913: T86_P1_F5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810913 GSM1810913 T86_P1_F5_ILC3 94071530 2187710 2016-01-28 01:00:06 102896902 94071530 2187710 1 2187710 index:0,count:2187710,average:43,stdev:0 GSM1810913_r1 7.16 6.64 0.22 60648958 78783336 48369452 64900446 129.9 134.18 0 0 0 0 0 0 59.78 76.06 2327649 881595 2327649 881595 66.98 72.87 2327649 987858 2327649 844699 8268419 13.63 6.96 0 14.43 0 1.13 0 0.33 0 0.00 0 31.13 0 1474748 0 43 0 41.73 0 1.13 0 0.01 0 1.12 0 0.00 0 231.64 0 0.37 0 152238 0 2187710 0 315630 0 24778 0 7194 0 0 0 680990 0 15 0 0 0 313 0 39686 0 733 0 40747 0 52.98 0 1159118 0 9588 46724 4.873174801836 2187710.0 1474748.0 152238.0 315630.0 24778.0 7194.0 0.0 680990.0 1159118.0 67.4 7.0 14.4 1.1 0.3 0.0 31.1 53.0 43 43 43.00 38 94071530 26.4 22.4 22.6 28.6 0.0 35.7 24.0 smartseq 1066905 SRR2088479 SRP060416 SRS979859 SRX1082448 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810914: T86_P1_F6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810914 GSM1810914 T86_P1_F6_ILC3 107528122 2500654 2016-01-28 01:00:06 117493274 107528122 2500654 1 2500654 index:0,count:2500654,average:43,stdev:0 GSM1810914_r1 11.54 6.34 0.19 72511141 98220405 57672137 80430736 135.46 139.46 0 0 0 0 0 0 60.97 77.65 2745819 1071281 2745819 1071281 68.88 74.11 2745819 1210241 2745819 1022502 9292351 12.82 6.36 0 15.09 0 0.98 0 0.39 0 0.00 0 28.37 0 1756931 0 43 0 41.80 0 1.09 0 0.01 0 1.11 0 0.00 0 290.40 0 0.37 0 158999 0 2500654 0 377231 0 24627 0 9777 0 0 0 709319 0 13 0 0 0 387 0 45963 0 760 0 47123 0 55.17 0 1379700 0 10432 53358 5.114838957055 2500654.0 1756931.0 158999.0 377231.0 24627.0 9777.0 0.0 709319.0 1379700.0 70.3 6.4 15.1 1.0 0.4 0.0 28.4 55.2 43 43 43.00 38 107528122 26.4 22.4 22.6 28.6 0.0 35.8 24.2 smartseq 1067017 SRR2088480 SRP060416 SRS979856 SRX1082449 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810915: T86_P1_F7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810915 GSM1810915 T86_P1_F7_ILC3 99312499 2309593 2016-01-28 01:00:06 108595409 99312499 2309593 1 2309593 index:0,count:2309593,average:43,stdev:0 GSM1810915_r1 10.99 6.74 0.22 64366563 86744899 51577267 71398954 134.77 138.43 0 0 0 0 0 0 62.85 79.52 2409992 982589 2409992 982589 69.94 75.6 2409992 1093529 2409992 934118 8029258 12.47 6.99 0 14.19 0 0.83 0 0.35 0 0.00 0 31.12 0 1563450 0 43 0 41.74 0 1.10 0 0.01 0 1.15 0 0.01 0 237.56 0 0.37 0 161513 0 2309593 0 327816 0 19236 0 8076 0 0 0 718831 0 43 0 0 0 360 0 44227 0 718 0 45348 0 53.50 0 1235634 0 9488 49860 5.255059021922 2309593.0 1563450.0 161513.0 327816.0 19236.0 8076.0 0.0 718831.0 1235634.0 67.7 7.0 14.2 0.8 0.3 0.0 31.1 53.5 43 43 43.00 38 99312499 26.4 22.5 22.6 28.5 0.0 35.7 24.1 smartseq 1067032 SRR2088481 SRP060416 SRS979855 SRX1082450 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810916: T86_P1_F8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810916 GSM1810916 T86_P1_F8_ILC3 56889430 1323010 2016-01-28 01:00:06 62645366 56889430 1323010 1 1323010 index:0,count:1323010,average:43,stdev:0 GSM1810916_r1 8.78 6.25 0.23 40184397 53501891 33113695 45164071 133.14 136.39 0 0 0 0 0 0 64.36 78.96 1427479 624805 1427479 624805 70.09 74.91 1427479 680432 1427479 592732 4606176 11.46 5.67 0 13.57 0 0.86 0 0.38 0 0.00 0 25.39 0 970769 0 43 0 41.85 0 1.17 0 0.01 0 1.12 0 0.00 0 264.60 0 0.37 0 74995 0 1323010 0 179486 0 11323 0 5062 0 0 0 335856 0 9 0 0 0 190 0 29422 0 413 0 30034 0 59.81 0 791283 0 10126 33066 3.265455263678 1323010.0 970769.0 74995.0 179486.0 11323.0 5062.0 0.0 335856.0 791283.0 73.4 5.7 13.6 0.9 0.4 0.0 25.4 59.8 43 43 43.00 38 56889430 26.4 22.7 22.9 28.1 0.0 35.7 24.2 smartseq 1067050 SRR2088482 SRP060416 SRS979853 SRX1082451 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810917: T86_P1_F9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810917 GSM1810917 T86_P1_F9_ILC3 19449244 452308 2016-01-28 01:00:06 21493521 19449244 452308 1 452308 index:0,count:452308,average:43,stdev:0 GSM1810917_r1 10.02 6.73 0.2 12568166 16791036 9964042 13722961 133.6 137.72 0 0 0 0 0 0 61.58 78.77 479977 187966 479977 187966 68.45 74.42 479977 208934 479977 177580 1618120 12.87 7.00 0 14.73 0 0.92 0 0.31 0 0.00 0 31.28 0 305246 0 43 0 41.76 0 1.10 0 0.01 0 1.12 0 0.00 0 85.70 0 0.37 0 31646 0 452308 0 66618 0 4158 0 1407 0 0 0 141497 0 6 0 0 0 78 0 8384 0 143 0 8611 0 52.76 0 238628 0 4852 9420 1.941467436109 452308.0 305246.0 31646.0 66618.0 4158.0 1407.0 0.0 141497.0 238628.0 67.5 7.0 14.7 0.9 0.3 0.0 31.3 52.8 43 43 43.00 38 19449244 26.5 22.5 22.6 28.5 0.0 35.8 24.1 smartseq 1067066 SRR2088483 SRP060416 SRS979852 SRX1082452 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810918: T86_P1_G10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810918 GSM1810918 T86_P1_G10_ILC3 70433914 1637998 2016-01-28 01:00:06 77463789 70433914 1637998 1 1637998 index:0,count:1637998,average:43,stdev:0 GSM1810918_r1 11.47 5.97 0.17 49401400 67217286 40093715 55768858 136.06 139.1 0 0 0 0 0 0 64.63 80.49 1779883 771124 1779883 771124 72.14 76.83 1779883 860770 1779883 736065 5672201 11.48 5.82 0 14.36 0 0.76 0 0.32 0 0.00 0 26.08 0 1193210 0 43 0 41.85 0 1.09 0 0.01 0 1.14 0 0.01 0 173.44 0 0.36 0 95406 0 1637998 0 235149 0 12431 0 5177 0 0 0 427180 0 27 0 0 0 222 0 36384 0 491 0 37124 0 58.49 0 958061 0 10491 41319 3.938518730340 1637998.0 1193210.0 95406.0 235149.0 12431.0 5177.0 0.0 427180.0 958061.0 72.8 5.8 14.4 0.8 0.3 0.0 26.1 58.5 43 43 43.00 38 70433914 26.5 22.5 22.7 28.2 0.0 35.8 24.4 smartseq 1067083 SRR2088484 SRP060416 SRS979857 SRX1082453 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810919: T86_P1_G12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810919 GSM1810919 T86_P1_G12_ILC3 145772150 3390050 2016-01-28 01:00:06 158412580 145772150 3390050 1 3390050 index:0,count:3390050,average:43,stdev:0 GSM1810919_r1 10.35 6.53 0.2 94676808 125599071 75596735 103146212 132.66 136.44 0 0 0 0 0 0 61.11 77.63 3564099 1404711 3564099 1404711 68.36 74.27 3564099 1571294 3564099 1343894 12231366 12.92 6.92 0 14.43 0 0.81 0 0.32 0 0.00 0 31.07 0 2298604 0 43 0 41.78 0 1.11 0 0.01 0 1.12 0 0.00 0 271.20 0 0.36 0 234483 0 3390050 0 489065 0 27539 0 10763 0 0 0 1053144 0 27 0 0 0 553 0 62285 0 1043 0 63908 0 53.38 0 1809539 0 11734 71487 6.092295892279 3390050.0 2298604.0 234483.0 489065.0 27539.0 10763.0 0.0 1053144.0 1809539.0 67.8 6.9 14.4 0.8 0.3 0.0 31.1 53.4 43 43 43.00 38 145772150 26.2 22.5 22.7 28.6 0.0 35.8 24.3 smartseq 1067098 SRR2088485 SRP060416 SRS979854 SRX1082454 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810920: T86_P1_G1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810920 GSM1810920 T86_P1_G1_ILC3 133631186 3107702 2016-01-28 01:00:06 145320290 133631186 3107702 1 3107702 index:0,count:3107702,average:43,stdev:0 GSM1810920_r1 11.34 6.53 0.17 88933320 119976377 72263159 100060180 134.91 138.47 0 0 0 0 0 0 62.19 77.51 3243593 1338229 3243593 1338229 68.86 74.1 3243593 1481727 3243593 1279335 11313096 12.72 6.63 0 13.69 0 0.83 0 0.36 0 0.00 0 29.56 0 2151864 0 43 0 41.86 0 1.14 0 0.01 0 1.11 0 0.00 0 243.21 0 0.35 0 206165 0 3107702 0 425434 0 25943 0 11282 0 0 0 918613 0 34 0 0 0 402 0 58310 0 954 0 59700 0 55.55 0 1726430 0 11842 67247 5.678686032765 3107702.0 2151864.0 206165.0 425434.0 25943.0 11282.0 0.0 918613.0 1726430.0 69.2 6.6 13.7 0.8 0.4 0.0 29.6 55.6 43 43 43.00 38 133631186 26.4 22.5 22.6 28.6 0.0 35.9 24.5 smartseq 1067114 SRR2088486 SRP060416 SRS979850 SRX1082455 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810921: T86_P1_G3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810921 GSM1810921 T86_P1_G3_ILC3 123201880 2865160 2016-01-28 01:00:06 133982912 123201880 2865160 1 2865160 index:0,count:2865160,average:43,stdev:0 GSM1810921_r1 10.59 7.58 0.23 72859757 95514050 57179104 77561176 131.09 135.65 0 0 0 0 0 0 59.51 77.22 2874676 1060488 2874676 1060488 66.07 73.81 2874676 1177380 2874676 1013622 10350950 14.21 8.19 0 14.26 0 0.90 0 0.30 0 0.00 0 36.60 0 1781952 0 43 0 41.63 0 1.10 0 0.01 0 1.09 0 0.00 0 245.59 0 0.37 0 234598 0 2865160 0 408605 0 25791 0 8732 0 0 0 1048685 0 25 0 0 0 353 0 44657 0 832 0 45867 0 47.93 0 1373347 0 8265 50359 6.093042952208 2865160.0 1781952.0 234598.0 408605.0 25791.0 8732.0 0.0 1048685.0 1373347.0 62.2 8.2 14.3 0.9 0.3 0.0 36.6 47.9 43 43 43.00 38 123201880 26.5 22.2 22.3 29.1 0.0 35.8 24.2 smartseq 1067131 SRR2088487 SRP060416 SRS979849 SRX1082456 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810922: T86_P1_G5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810922 GSM1810922 T86_P1_G5_ILC3 109700998 2551186 2016-01-28 01:00:06 119901608 109700998 2551186 1 2551186 index:0,count:2551186,average:43,stdev:0 GSM1810922_r1 7.94 6.94 0.21 72211856 94596859 57943139 78297361 131.0 135.13 0 0 0 0 0 0 60.24 76.17 2722463 1056361 2722463 1056361 66.87 72.66 2722463 1172587 2722463 1007750 9737429 13.48 6.71 0 14.38 0 1.05 0 0.34 0 0.00 0 29.87 0 1753639 0 43 0 41.78 0 1.18 0 0.01 0 1.13 0 0.01 0 306.14 0 0.36 0 171074 0 2551186 0 366759 0 26825 0 8642 0 0 0 762080 0 19 0 0 0 362 0 48885 0 838 0 50104 0 54.36 0 1386880 0 11124 56707 5.097716648688 2551186.0 1753639.0 171074.0 366759.0 26825.0 8642.0 0.0 762080.0 1386880.0 68.7 6.7 14.4 1.1 0.3 0.0 29.9 54.4 43 43 43.00 38 109700998 26.4 22.3 22.5 28.7 0.0 35.7 24.1 smartseq 1067147 SRR2088488 SRP060416 SRS979848 SRX1082457 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810923: T86_P1_G6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810923 GSM1810923 T86_P1_G6_ILC3 129954127 3022189 2016-01-28 01:00:06 142282475 129954127 3022189 1 3022189 index:0,count:3022189,average:43,stdev:0 GSM1810923_r1 8.5 6.82 0.21 85101858 111708302 66764856 90525968 131.26 135.59 0 0 0 0 0 0 57.72 74.7 3308101 1194707 3308101 1194707 64.93 70.73 3308101 1344063 3308101 1131131 11770613 13.83 6.71 0 15.57 0 1.19 0 0.42 0 0.00 0 29.90 0 2069876 0 43 0 41.75 0 1.16 0 0.01 0 1.14 0 0.00 0 226.66 0 0.37 0 202793 0 3022189 0 470594 0 36114 0 12565 0 0 0 903634 0 12 0 0 0 426 0 54110 0 979 0 55527 0 52.92 0 1599282 0 11427 64413 5.636912575479 3022189.0 2069876.0 202793.0 470594.0 36114.0 12565.0 0.0 903634.0 1599282.0 68.5 6.7 15.6 1.2 0.4 0.0 29.9 52.9 43 43 43.00 38 129954127 26.1 22.3 22.5 29.0 0.0 35.6 24.0 smartseq 1067163 SRR2088489 SRP060416 SRS979847 SRX1082458 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810924: T86_P1_G7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810924 GSM1810924 T86_P1_G7_ILC3 126842518 2949826 2016-01-28 01:00:06 139232696 126842518 2949826 1 2949826 index:0,count:2949826,average:43,stdev:0 GSM1810924_r1 9.39 6.22 0.21 86422914 115319880 69078509 94887921 133.44 137.36 0 0 0 0 0 0 60.93 77.19 3254499 1274729 3254499 1274729 68.03 73.16 3254499 1423239 3254499 1208122 10695317 12.38 6.17 0 14.94 0 1.10 0 0.38 0 0.00 0 27.60 0 2091978 0 43 0 41.83 0 1.13 0 0.01 0 1.10 0 0.00 0 259.01 0 0.38 0 181937 0 2949826 0 440620 0 32487 0 11261 0 0 0 814100 0 47 0 0 0 432 0 56299 0 851 0 57629 0 55.98 0 1651358 0 11211 65097 5.806529301579 2949826.0 2091978.0 181937.0 440620.0 32487.0 11261.0 0.0 814100.0 1651358.0 70.9 6.2 14.9 1.1 0.4 0.0 27.6 56.0 43 43 43.00 38 126842518 26.3 22.6 22.8 28.3 0.0 35.6 24.1 smartseq 1067275 SRR2088490 SRP060416 SRS979851 SRX1082459 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810925: T86_P1_G8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810925 GSM1810925 T86_P1_G8_ILC3 77706676 1807132 2016-01-28 01:00:06 85293187 77706676 1807132 1 1807132 index:0,count:1807132,average:43,stdev:0 GSM1810925_r1 10.86 5.52 0.17 55287233 73645848 45372421 61551870 133.21 135.66 0 0 0 0 0 0 62.4 76.8 1964353 830983 1964353 830983 69.23 73.23 1964353 921992 1964353 792293 7073043 12.79 5.64 0 13.82 0 0.70 0 0.37 0 0.00 0 25.24 0 1331721 0 43 0 41.94 0 1.14 0 0.01 0 1.13 0 0.00 0 260.23 0 0.36 0 101992 0 1807132 0 249772 0 12673 0 6706 0 0 0 456032 0 15 0 0 0 263 0 38110 0 601 0 38989 0 59.87 0 1081949 0 10798 43766 4.053157992221 1807132.0 1331721.0 101992.0 249772.0 12673.0 6706.0 0.0 456032.0 1081949.0 73.7 5.6 13.8 0.7 0.4 0.0 25.2 59.9 43 43 43.00 38 77706676 26.5 22.6 22.8 28.1 0.0 35.8 24.5 smartseq 1067290 SRR2088491 SRP060416 SRS979846 SRX1082460 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810926: T86_P1_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810926 GSM1810926 T86_P1_G9_NK 26000294 604658 2016-01-28 01:00:06 28707414 26000294 604658 1 604658 index:0,count:604658,average:43,stdev:0 GSM1810926_r1 11.95 6.19 0.21 17579248 24380668 13943691 19963530 138.69 143.17 0 0 0 0 0 0 63.75 81.33 666496 270911 666496 270911 72.37 78.05 666496 307565 666496 260007 1885506 10.73 6.41 0 15.19 0 0.86 0 0.32 0 0.00 0 28.53 0 424970 0 43 0 41.86 0 1.14 0 0.01 0 1.10 0 0.01 0 136.05 0 0.37 0 38730 0 604658 0 91861 0 5220 0 1960 0 0 0 172508 0 7 0 0 0 98 0 11235 0 177 0 11517 0 55.09 0 333109 0 5345 12700 2.376052385407 604658.0 424970.0 38730.0 91861.0 5220.0 1960.0 0.0 172508.0 333109.0 70.3 6.4 15.2 0.9 0.3 0.0 28.5 55.1 43 43 43.00 38 26000294 26.4 22.6 22.7 28.3 0.0 35.8 24.4 smartseq 1067306 SRR2088492 SRP060416 SRS979845 SRX1082461 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810927: T86_P1_H10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810927 GSM1810927 T86_P1_H10_ILC3 23081884 536788 2016-01-28 01:00:06 26444627 23081884 536788 1 536788 index:0,count:536788,average:43,stdev:0 GSM1810927_r1 10.55 6.75 0.18 15724454 20581837 12681044 17036287 130.89 134.34 0 0 0 0 0 0 59.45 74.81 586209 227488 586209 227488 65.88 71.48 586209 252076 586209 217350 2339032 14.88 6.12 0 14.64 0 0.88 0 0.43 0 0.00 0 27.41 0 382636 0 43 0 41.70 0 1.18 0 0.01 0 1.09 0 0.00 0 128.83 0 0.46 0 32832 0 536788 0 78561 0 4713 0 2301 0 0 0 147138 0 3 0 0 0 72 0 9808 0 155 0 10038 0 56.65 0 304075 0 5498 10559 1.920516551473 536788.0 382636.0 32832.0 78561.0 4713.0 2301.0 0.0 147138.0 304075.0 71.3 6.1 14.6 0.9 0.4 0.0 27.4 56.6 43 43 43.00 38 23081884 27.2 21.4 22.4 29.0 0.0 34.7 22.8 smartseq 1067321 SRR2088493 SRP060416 SRS979843 SRX1082462 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810928: T86_P1_H11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810928 GSM1810928 T86_P1_H11_ILC3 104458094 2429258 2016-01-28 01:00:06 114633175 104458094 2429258 1 2429258 index:0,count:2429258,average:43,stdev:0 GSM1810928_r1 10.41 6.96 0.28 65934464 87090368 51780046 70537416 132.09 136.23 0 0 0 0 0 0 60.63 78.43 2572050 974891 2572050 974891 68.61 75.41 2572050 1103308 2572050 937458 8519250 12.92 7.30 0 15.02 0 0.84 0 0.28 0 0.00 0 32.68 0 1608057 0 43 0 41.66 0 1.12 0 0.01 0 1.14 0 0.00 0 301.56 0 0.39 0 177267 0 2429258 0 364989 0 20399 0 6829 0 0 0 793973 0 17 0 0 0 254 0 39739 0 746 0 40756 0 51.17 0 1243068 0 8291 45303 5.464117718007 2429258.0 1608057.0 177267.0 364989.0 20399.0 6829.0 0.0 793973.0 1243068.0 66.2 7.3 15.0 0.8 0.3 0.0 32.7 51.2 43 43 43.00 38 104458094 26.3 22.4 22.5 28.8 0.0 35.6 23.9 smartseq 1067339 SRR2088494 SRP060416 SRS979842 SRX1082463 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810929: T86_P1_H12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810929 GSM1810929 T86_P1_H12_ILC3 103572853 2408671 2016-01-28 01:00:06 113417395 103572853 2408671 1 2408671 index:0,count:2408671,average:43,stdev:0 GSM1810929_r1 10.17 6.51 0.19 70403367 93463960 56995714 77822680 132.75 136.54 0 0 0 0 0 0 60.66 75.96 2606399 1037342 2606399 1037342 67.04 72.43 2606399 1146429 2606399 989046 10354348 14.71 6.16 0 14.31 0 0.96 0 0.43 0 0.00 0 27.62 0 1710193 0 43 0 41.74 0 1.13 0 0.01 0 1.14 0 0.00 0 270.98 0 0.38 0 148358 0 2408671 0 344597 0 23009 0 10310 0 0 0 665159 0 19 0 0 0 289 0 42298 0 772 0 43378 0 56.69 0 1365596 0 9410 47619 5.060467587673 2408671.0 1710193.0 148358.0 344597.0 23009.0 10310.0 0.0 665159.0 1365596.0 71.0 6.2 14.3 1.0 0.4 0.0 27.6 56.7 43 43 43.00 38 103572853 26.7 22.2 22.4 28.7 0.0 35.7 24.1 smartseq 1067354 SRR2088495 SRP060416 SRS979844 SRX1082464 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810930: T86_P1_H2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810930 GSM1810930 T86_P1_H2_ILC3 44668013 1038791 2016-01-28 01:00:06 50987591 44668013 1038791 1 1038791 index:0,count:1038791,average:43,stdev:0 GSM1810930_r1 11.76 7.87 0.19 27857783 35959848 21605725 28794856 129.08 133.27 0 0 0 0 0 0 55.14 72.42 1110781 375960 1110781 375960 62.61 69.23 1110781 426906 1110781 359378 4453609 15.99 7.46 0 15.67 0 0.85 0 0.36 0 0.00 0 33.15 0 681859 0 43 0 41.62 0 1.15 0 0.01 0 1.11 0 0.00 0 233.73 0 0.47 0 77493 0 1038791 0 162754 0 8870 0 3694 0 0 0 344368 0 3 0 0 0 114 0 14724 0 296 0 15137 0 49.97 0 519105 0 6166 16580 2.688939344794 1038791.0 681859.0 77493.0 162754.0 8870.0 3694.0 0.0 344368.0 519105.0 65.6 7.5 15.7 0.9 0.4 0.0 33.2 50.0 43 43 43.00 38 44668013 26.8 21.3 22.4 29.5 0.0 34.6 22.7 smartseq 1067371 SRR2088496 SRP060416 SRS979841 SRX1082465 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810931: T86_P1_H3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810931 GSM1810931 T86_P1_H3_ILC3 103289870 2402090 2016-01-28 01:00:06 113374879 103289870 2402090 1 2402090 index:0,count:2402090,average:43,stdev:0 GSM1810931_r1 6.98 7.21 0.23 64455070 81510304 49977375 65329886 126.46 130.72 0 0 0 0 0 0 54.0 70.82 2605848 849789 2605848 849789 61.26 67.05 2605848 963962 2605848 804520 10748370 16.68 7.36 0 15.55 0 1.07 0 0.44 0 0.00 0 32.98 0 1573560 0 43 0 41.65 0 1.18 0 0.01 0 1.12 0 0.00 0 254.34 0 0.39 0 176892 0 2402090 0 373601 0 25674 0 10562 0 0 0 792294 0 13 0 0 0 388 0 38162 0 839 0 39402 0 49.95 0 1199959 0 7880 44893 5.697081218274 2402090.0 1573560.0 176892.0 373601.0 25674.0 10562.0 0.0 792294.0 1199959.0 65.5 7.4 15.6 1.1 0.4 0.0 33.0 50.0 43 43 43.00 38 103289870 26.4 22.2 22.4 29.0 0.0 35.6 23.9 smartseq 1067387 SRR2088497 SRP060416 SRS979840 SRX1082466 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810932: T86_P1_H4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810932 GSM1810932 T86_P1_H4_ILC3 96577484 2245988 2016-01-28 01:00:06 105919497 96577484 2245988 1 2245988 index:0,count:2245988,average:43,stdev:0 GSM1810932_r1 8.68 6.93 0.23 62645912 82651611 50570193 68705512 131.93 135.86 0 0 0 0 0 0 59.64 74.97 2325079 906553 2325079 906553 66.24 71.56 2325079 1006856 2325079 865367 8107269 12.94 6.97 0 13.83 0 0.87 0 0.30 0 0.00 0 31.15 0 1519917 0 43 0 41.82 0 1.14 0 0.01 0 1.16 0 0.01 0 224.60 0 0.38 0 156567 0 2245988 0 310705 0 19569 0 6838 0 0 0 699664 0 10 0 0 0 303 0 39169 0 732 0 40214 0 53.84 0 1209212 0 9452 45174 4.779305966991 2245988.0 1519917.0 156567.0 310705.0 19569.0 6838.0 0.0 699664.0 1209212.0 67.7 7.0 13.8 0.9 0.3 0.0 31.2 53.8 43 43 43.00 38 96577484 26.2 22.4 22.6 28.7 0.0 35.7 24.0 smartseq 1067401 SRR2088498 SRP060416 SRS979839 SRX1082467 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810933: T86_P1_H5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810933 GSM1810933 T86_P1_H5_ILC3 112924880 2626160 2016-01-28 01:00:06 123508836 112924880 2626160 1 2626160 index:0,count:2626160,average:43,stdev:0 GSM1810933_r1 8.06 7.89 0.22 66435953 84774851 50893349 67577140 127.6 132.78 0 0 0 0 0 0 56.77 75.58 2743045 925716 2743045 925716 63.97 72.11 2743045 1043180 2743045 883202 10199837 15.35 8.22 0 15.46 0 0.95 0 0.31 0 0.00 0 36.65 0 1630772 0 43 0 41.55 0 1.15 0 0.01 0 1.12 0 0.00 0 262.62 0 0.39 0 215824 0 2626160 0 405984 0 24913 0 8031 0 0 0 962444 0 8 0 0 0 356 0 41023 0 941 0 42328 0 46.64 0 1224788 0 7472 46892 6.275695931478 2626160.0 1630772.0 215824.0 405984.0 24913.0 8031.0 0.0 962444.0 1224788.0 62.1 8.2 15.5 0.9 0.3 0.0 36.6 46.6 43 43 43.00 38 112924880 26.3 22.1 22.3 29.2 0.0 35.6 23.7 smartseq 1067417 SRR2088499 SRP060416 SRS979838 SRX1082468 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810934: T86_P1_H8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810934 GSM1810934 T86_P1_H8_ILC3 75616790 1758530 2016-01-28 01:00:06 83471021 75616790 1758530 1 1758530 index:0,count:1758530,average:43,stdev:0 GSM1810934_r1 10.92 6.55 0.18 52941348 71795001 42604101 59308996 135.61 139.21 0 0 0 0 0 0 62.0 77.95 1960973 793610 1960973 793610 69.52 74.4 1960973 889984 1960973 757478 6478118 12.24 5.83 0 14.90 0 0.79 0 0.33 0 0.00 0 26.09 0 1280107 0 43 0 41.85 0 1.12 0 0.01 0 1.11 0 0.00 0 211.02 0 0.39 0 102437 0 1758530 0 262033 0 13847 0 5757 0 0 0 458819 0 16 0 0 0 281 0 34618 0 540 0 35455 0 57.89 0 1018074 0 9938 39326 3.957134232240 1758530.0 1280107.0 102437.0 262033.0 13847.0 5757.0 0.0 458819.0 1018074.0 72.8 5.8 14.9 0.8 0.3 0.0 26.1 57.9 43 43 43.00 38 75616790 26.5 22.5 22.7 28.3 0.0 35.6 24.0 smartseq 1069065 SRR2088500 SRP060416 SRS979836 SRX1082469 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810935: T86_P1_H9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810935 GSM1810935 T86_P1_H9_ILC3 24275220 564540 2016-01-28 01:00:06 26841386 24275220 564540 1 564540 index:0,count:564540,average:43,stdev:0 GSM1810935_r1 6.94 6.78 0.23 15216518 19650175 12095744 16153325 129.14 133.55 0 0 0 0 0 0 58.92 75.37 589994 218602 589994 218602 65.64 72.03 589994 243535 589994 208919 2158765 14.19 7.38 0 14.35 0 1.04 0 0.40 0 0.00 0 32.83 0 371033 0 43 0 41.71 0 1.14 0 0.01 0 1.10 0 0.00 0 112.91 0 0.38 0 41651 0 564540 0 81006 0 5896 0 2272 0 0 0 185339 0 4 0 0 0 72 0 9416 0 184 0 9676 0 51.37 0 290027 0 4785 10532 2.201044932079 564540.0 371033.0 41651.0 81006.0 5896.0 2272.0 0.0 185339.0 290027.0 65.7 7.4 14.3 1.0 0.4 0.0 32.8 51.4 43 43 43.00 38 24275220 26.5 22.2 22.4 28.9 0.0 35.7 23.9 smartseq 1069081 SRR2088501 SRP060416 SRS979837 SRX1082470 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810936: T86_P2_A10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810936 GSM1810936 T86_P2_A10_ILC2 19272557 448199 2016-01-28 01:00:06 21934028 19272557 448199 1 448199 index:0,count:448199,average:43,stdev:0 GSM1810936_r1 4.43 8.47 0.2 12912023 16039124 9715783 12541617 124.22 129.08 0 0 0 0 0 0 55.45 75.25 552880 176992 552880 176992 63.33 71.63 552880 202158 552880 168493 1958578 15.17 5.93 0 18.74 0 1.34 0 0.38 0 0.00 0 27.06 0 319192 0 43 0 41.31 0 1.22 0 0.00 0 1.14 0 0.00 0 89.64 0 0.45 0 26565 0 448199 0 83974 0 5992 0 1723 0 0 0 121292 0 6 0 0 0 50 0 7666 0 180 0 7902 0 52.48 0 235218 0 3738 8556 2.288924558587 448199.0 319192.0 26565.0 83974.0 5992.0 1723.0 0.0 121292.0 235218.0 71.2 5.9 18.7 1.3 0.4 0.0 27.1 52.5 43 43 43.00 38 19272557 27.3 20.9 21.7 30.1 0.0 35.0 23.0 smartseq 1069098 SRR2088502 SRP060416 SRS979835 SRX1082471 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810937: T86_P2_A12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810937 GSM1810937 T86_P2_A12_ILC2 52800130 1227910 2016-01-28 01:00:06 59989485 52800130 1227910 1 1227910 index:0,count:1227910,average:43,stdev:0 GSM1810937_r1 3.63 9.25 0.23 35142085 43544802 26752592 34700656 123.91 129.71 0 0 0 0 0 0 55.1 74.11 1481485 481177 1481485 481177 61.43 71.0 1481485 536435 1481485 460977 5851657 16.65 6.18 0 18.24 0 1.20 0 0.41 0 0.00 0 27.28 0 873225 0 43 0 41.21 0 1.28 0 0.01 0 1.15 0 0.00 0 232.66 0 0.45 0 75889 0 1227910 0 223976 0 14674 0 5069 0 0 0 334942 0 8 0 0 0 175 0 20687 0 495 0 21365 0 52.87 0 649249 0 5750 23262 4.045565217391 1227910.0 873225.0 75889.0 223976.0 14674.0 5069.0 0.0 334942.0 649249.0 71.1 6.2 18.2 1.2 0.4 0.0 27.3 52.9 43 43 43.00 38 52800130 27.5 20.4 21.2 30.9 0.0 34.9 22.7 smartseq 1069113 SRR2088503 SRP060416 SRS979834 SRX1082472 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810938: T86_P2_A3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810938 GSM1810938 T86_P2_A3_ILC2 126020831 2930717 2016-01-28 01:00:06 135954936 126020831 2930717 1 2930717 index:0,count:2930717,average:43,stdev:0 GSM1810938_r1 3.49 7.83 0.25 90301453 115067995 70600702 92776326 127.43 131.41 0 0 0 0 0 0 59.32 77.11 3550461 1308610 3550461 1308610 66.54 73.02 3550461 1467829 3550461 1239256 12423493 13.76 5.10 0 17.37 0 1.23 0 0.51 0 0.00 0 22.99 0 2206099 0 43 0 41.60 0 1.21 0 0.01 0 1.16 0 0.00 0 405.79 0 0.36 0 149509 0 2930717 0 508964 0 36010 0 14977 0 0 0 673631 0 59 0 0 0 575 0 63814 0 1147 0 65595 0 57.91 0 1697135 0 8684 76432 8.801473975127 2930717.0 2206099.0 149509.0 508964.0 36010.0 14977.0 0.0 673631.0 1697135.0 75.3 5.1 17.4 1.2 0.5 0.0 23.0 57.9 43 43 43.00 38 126020831 26.9 21.8 21.9 29.4 0.0 36.2 24.6 smartseq 1069129 SRR2088504 SRP060416 SRS979833 SRX1082473 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810939: T86_P2_A5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810939 GSM1810939 T86_P2_A5_ILC2 57477025 1336675 2016-01-28 01:00:06 63261793 57477025 1336675 1 1336675 index:0,count:1336675,average:43,stdev:0 GSM1810939_r1 4.38 6.74 0.25 44738347 58871823 36250410 48843291 131.59 134.74 0 0 0 0 0 0 63.66 79.5 1640003 692592 1640003 692592 70.19 75.51 1640003 763638 1640003 657867 5234048 11.70 3.71 0 16.22 0 1.08 0 0.49 0 0.00 0 17.03 0 1088003 0 43 0 41.61 0 1.24 0 0.01 0 1.12 0 0.00 0 283.06 0 0.36 0 49649 0 1336675 0 216783 0 14490 0 6573 0 0 0 227609 0 5 0 0 0 189 0 32333 0 639 0 33166 0 65.18 0 871220 0 8964 38019 4.241298527443 1336675.0 1088003.0 49649.0 216783.0 14490.0 6573.0 0.0 227609.0 871220.0 81.4 3.7 16.2 1.1 0.5 0.0 17.0 65.2 43 43 43.00 38 57477025 27.2 21.7 21.9 29.2 0.0 36.0 24.6 smartseq 1069146 SRR2088505 SRP060416 SRS979830 SRX1082474 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810940: T86_P2_A7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810940 GSM1810940 T86_P2_A7_ILC2 40257116 936212 2016-01-28 01:00:06 46028995 40257116 936212 1 936212 index:0,count:936212,average:43,stdev:0 GSM1810940_r1 5.24 8.91 0.21 27944495 35180821 21861478 28594951 125.9 130.8 0 0 0 0 0 0 57.93 75.55 1119273 399479 1119273 399479 64.53 72.66 1119273 445021 1119273 384197 4164170 14.90 5.63 0 17.18 0 1.25 0 0.39 0 0.00 0 24.71 0 689619 0 43 0 41.35 0 1.23 0 0.01 0 1.14 0 0.01 0 210.65 0 0.46 0 52707 0 936212 0 160882 0 11681 0 3607 0 0 0 231305 0 14 0 0 0 118 0 14639 0 383 0 15154 0 56.48 0 528737 0 4872 16548 3.396551724138 936212.0 689619.0 52707.0 160882.0 11681.0 3607.0 0.0 231305.0 528737.0 73.7 5.6 17.2 1.2 0.4 0.0 24.7 56.5 43 43 43.00 38 40257116 27.5 20.5 21.7 30.3 0.0 34.8 22.7 smartseq 1069165 SRR2088506 SRP060416 SRS979781 SRX1082475 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810941: T86_P2_A8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810941 GSM1810941 T86_P2_A8_ILC2 35053858 815206 2016-01-28 01:00:06 38797215 35053858 815206 1 815206 index:0,count:815206,average:43,stdev:0 GSM1810941_r1 5.59 7.43 0.28 24790665 32260322 19430022 26154354 130.13 134.61 0 0 0 0 0 0 61.69 80.07 982924 375285 982924 375285 68.84 76.22 982924 418766 982924 357243 3221642 13.00 5.33 0 17.13 0 1.14 0 0.40 0 0.00 0 23.84 0 608342 0 43 0 41.46 0 1.21 0 0.01 0 1.14 0 0.00 0 183.42 0 0.39 0 43478 0 815206 0 139660 0 9284 0 3223 0 0 0 194357 0 1 0 0 0 87 0 15744 0 361 0 16193 0 57.49 0 468682 0 5279 18027 3.414851297594 815206.0 608342.0 43478.0 139660.0 9284.0 3223.0 0.0 194357.0 468682.0 74.6 5.3 17.1 1.1 0.4 0.0 23.8 57.5 43 43 43.00 38 35053858 27.2 21.5 21.6 29.7 0.0 35.8 24.0 smartseq 1069181 SRR2088507 SRP060416 SRS979831 SRX1082476 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810942: T86_P2_A9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810942 GSM1810942 T86_P2_A9_ILC2 27067382 629474 2016-01-28 01:00:06 29365335 27067382 629474 1 629474 index:0,count:629474,average:43,stdev:0 GSM1810942_r1 6.47 5.99 0.17 21696290 28768379 17205480 23206505 132.6 134.88 0 0 0 0 0 0 61.47 78.22 812221 321832 812221 321832 70.37 73.87 812221 368438 812221 303919 2513027 11.58 3.37 0 17.81 0 1.10 0 0.56 0 0.00 0 15.17 0 523564 0 43 0 41.82 0 1.22 0 0.01 0 1.11 0 0.00 0 161.86 0 0.34 0 21187 0 629474 0 112137 0 6896 0 3530 0 0 0 95484 0 6 0 0 0 117 0 14699 0 246 0 15068 0 65.36 0 411427 0 6521 17460 2.677503450391 629474.0 523564.0 21187.0 112137.0 6896.0 3530.0 0.0 95484.0 411427.0 83.2 3.4 17.8 1.1 0.6 0.0 15.2 65.4 43 43 43.00 38 27067382 26.7 22.3 22.3 28.7 0.0 36.5 25.4 smartseq 1069197 SRR2088508 SRP060416 SRS979832 SRX1082477 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810943: T86_P2_B12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810943 GSM1810943 T86_P2_B12_ILC2 218626792 5084344 2016-01-28 01:00:06 232896368 218626792 5084344 1 5084344 index:0,count:5084344,average:43,stdev:0 GSM1810943_r1 6.02 7.71 0.21 143285337 191013154 111366047 153426949 133.31 137.77 0 0 0 0 0 0 62.26 81.41 5659953 2181576 5659953 2181576 70.25 77.43 5659953 2461508 5659953 2074866 16466788 11.49 6.68 0 16.21 0 0.99 0 0.32 0 0.00 0 29.77 0 3504022 0 43 0 41.56 0 1.17 0 0.01 0 1.12 0 0.00 0 250.73 0 0.36 0 339886 0 5084344 0 824233 0 50557 0 16048 0 0 0 1513717 0 71 0 0 0 589 0 100889 0 1744 0 103293 0 52.71 0 2679789 0 9982 119772 11.998797836105 5084344.0 3504022.0 339886.0 824233.0 50557.0 16048.0 0.0 1513717.0 2679789.0 68.9 6.7 16.2 1.0 0.3 0.0 29.8 52.7 43 43 43.00 38 218626792 26.7 22.0 22.1 29.2 0.0 36.2 24.8 smartseq 1069213 SRR2088509 SRP060416 SRS979829 SRX1082478 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810944: T86_P2_B1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810944 GSM1810944 T86_P2_B1_ILC2 260320667 6053969 2016-01-28 01:00:06 274731225 260320667 6053969 1 6053969 index:0,count:6053969,average:43,stdev:0 GSM1810944_r1 5.63 8.08 0.21 161835549 213450380 128038338 174558644 131.89 136.33 0 0 0 0 0 0 63.27 81.39 6268234 2507313 6268234 2507313 69.87 77.64 6268234 2768827 6268234 2391788 20255097 12.52 7.53 0 14.57 0 0.95 0 0.29 0 0.00 0 33.30 0 3962766 0 43 0 41.56 0 1.19 0 0.01 0 1.12 0 0.00 0 247.66 0 0.35 0 455829 0 6053969 0 882009 0 57699 0 17262 0 0 0 2016242 0 30 0 0 0 770 0 110149 0 2255 0 113204 0 50.89 0 3080757 0 9833 129689 13.189158954541 6053969.0 3962766.0 455829.0 882009.0 57699.0 17262.0 0.0 2016242.0 3080757.0 65.5 7.5 14.6 1.0 0.3 0.0 33.3 50.9 43 43 43.00 38 260320667 26.6 21.9 22.0 29.4 0.0 36.3 24.8 smartseq 1069325 SRR2088510 SRP060416 SRS979828 SRX1082479 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810945: T86_P2_B3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810945 GSM1810945 T86_P2_B3_ILC2 175696280 4085960 2016-01-28 01:00:06 187232415 175696280 4085960 1 4085960 index:0,count:4085960,average:43,stdev:0 GSM1810945_r1 2.8 9.34 0.26 97728702 119923139 73157907 94021089 122.71 128.52 0 0 0 0 0 0 52.48 71.89 4245969 1276437 4245969 1276437 58.63 67.81 4245969 1425876 4245969 1203968 18754983 19.19 8.76 0 16.07 0 1.18 0 0.33 0 0.00 0 38.96 0 2432079 0 43 0 41.20 0 1.25 0 0.01 0 1.13 0 0.01 0 350.23 0 0.38 0 357774 0 4085960 0 656526 0 48321 0 13511 0 0 0 1592049 0 14 0 0 0 516 0 58323 0 1570 0 60423 0 43.45 0 1775553 0 6855 69728 10.171845368344 4085960.0 2432079.0 357774.0 656526.0 48321.0 13511.0 0.0 1592049.0 1775553.0 59.5 8.8 16.1 1.2 0.3 0.0 39.0 43.5 43 43 43.00 38 175696280 27.2 21.1 21.2 30.5 0.0 36.2 24.5 smartseq 1069340 SRR2088511 SRP060416 SRS979827 SRX1082480 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810946: T86_P2_B5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810946 GSM1810946 T86_P2_B5_ILC2 97655279 2271053 2016-01-28 01:00:06 105003685 97655279 2271053 1 2271053 index:0,count:2271053,average:43,stdev:0 GSM1810946_r1 3.72 7.24 0.23 62866492 80351587 49575441 65222917 127.81 131.56 0 0 0 0 0 0 61.72 79.54 2462422 952028 2462422 952028 68.57 75.34 2462422 1057609 2462422 901727 7977053 12.69 6.93 0 15.22 0 1.02 0 0.31 0 0.00 0 30.75 0 1542487 0 43 0 41.42 0 1.21 0 0.01 0 1.15 0 0.00 0 255.49 0 0.34 0 157371 0 2271053 0 345554 0 23218 0 7083 0 0 0 698265 0 10 0 0 0 267 0 40905 0 945 0 42127 0 52.70 0 1196933 0 6951 48339 6.954251186880 2271053.0 1542487.0 157371.0 345554.0 23218.0 7083.0 0.0 698265.0 1196933.0 67.9 6.9 15.2 1.0 0.3 0.0 30.7 52.7 43 43 43.00 38 97655279 27.1 21.6 21.5 29.7 0.0 36.3 24.9 smartseq 1069357 SRR2088512 SRP060416 SRS979826 SRX1082481 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810947: T86_P2_B6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810947 GSM1810947 T86_P2_B6_ILC2 124409922 2893254 2016-01-28 01:00:06 135527555 124409922 2893254 1 2893254 index:0,count:2893254,average:43,stdev:0 GSM1810947_r1 2.27 8.24 0.23 73842622 90014879 58203137 73613576 121.9 126.48 0 0 0 0 0 0 58.35 75.6 2962086 1064365 2962086 1064365 63.46 72.0 2962086 1157548 2962086 1013741 11550632 15.64 8.04 0 14.38 0 1.03 0 0.34 0 0.00 0 35.59 0 1824129 0 43 0 41.34 0 1.29 0 0.01 0 1.12 0 0.00 0 315.63 0 0.38 0 232652 0 2893254 0 416173 0 29718 0 9705 0 0 0 1029702 0 15 0 0 0 315 0 44032 0 1220 0 45582 0 48.66 0 1407956 0 6410 50828 7.929485179407 2893254.0 1824129.0 232652.0 416173.0 29718.0 9705.0 0.0 1029702.0 1407956.0 63.0 8.0 14.4 1.0 0.3 0.0 35.6 48.7 43 43 43.00 38 124409922 27.3 21.2 21.3 30.1 0.0 35.9 24.2 smartseq 1069374 SRR2088513 SRP060416 SRS979825 SRX1082482 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810948: T86_P2_B7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810948 GSM1810948 T86_P2_B7_ILC2 147129703 3421621 2016-01-28 01:00:06 157316728 147129703 3421621 1 3421621 index:0,count:3421621,average:43,stdev:0 GSM1810948_r1 3.93 7.25 0.21 97353042 126635580 77997381 104647095 130.08 134.17 0 0 0 0 0 0 62.47 79.13 3711725 1486962 3711725 1486962 68.74 75.09 3711725 1636105 3711725 1411052 12235024 12.57 6.58 0 14.64 0 0.97 0 0.39 0 0.00 0 29.08 0 2380181 0 43 0 41.51 0 1.27 0 0.01 0 1.13 0 0.00 0 342.16 0 0.35 0 225075 0 3421621 0 501077 0 33346 0 13185 0 0 0 994909 0 34 0 0 0 541 0 68720 0 1333 0 70628 0 54.92 0 1879104 0 10599 81238 7.664685347674 3421621.0 2380181.0 225075.0 501077.0 33346.0 13185.0 0.0 994909.0 1879104.0 69.6 6.6 14.6 1.0 0.4 0.0 29.1 54.9 43 43 43.00 38 147129703 26.8 22.0 21.9 29.3 0.0 36.4 25.0 smartseq 1069390 SRR2088514 SRP060416 SRS979824 SRX1082483 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810949: T86_P2_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810949 GSM1810949 T86_P2_B9_NK 35610321 828147 2016-01-28 01:00:06 38568145 35610321 828147 1 828147 index:0,count:828147,average:43,stdev:0 GSM1810949_r1 5.75 6.52 0.18 24880946 33340197 20029528 27661609 134.0 138.1 0 0 0 0 0 0 64.49 81.09 930224 389852 930224 389852 70.97 76.67 930224 429021 930224 368560 2726936 10.96 5.83 0 14.95 0 1.03 0 0.36 0 0.00 0 25.62 0 604507 0 43 0 41.66 0 1.27 0 0.01 0 1.15 0 0.00 0 198.76 0 0.34 0 48263 0 828147 0 123769 0 8496 0 3000 0 0 0 212144 0 3 0 0 0 118 0 18481 0 288 0 18890 0 58.05 0 480738 0 7780 21495 2.762853470437 828147.0 604507.0 48263.0 123769.0 8496.0 3000.0 0.0 212144.0 480738.0 73.0 5.8 14.9 1.0 0.4 0.0 25.6 58.0 43 43 43.00 38 35610321 26.8 22.0 22.0 29.2 0.0 36.4 25.2 smartseq 1069405 SRR2088515 SRP060416 SRS979823 SRX1082484 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810950: T86_P2_C10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810950 GSM1810950 T86_P2_C10_ILC2 38196943 888301 2016-01-28 01:00:06 42247283 38196943 888301 1 888301 index:0,count:888301,average:43,stdev:0 GSM1810950_r1 5.47 7.54 0.2 25719435 33138610 19965747 26669330 128.85 133.58 0 0 0 0 0 0 59.01 77.47 1040891 373447 1040891 373447 66.3 74.05 1040891 419587 1040891 356970 3546069 13.79 6.05 0 16.97 0 1.25 0 0.33 0 0.00 0 27.18 0 632847 0 43 0 41.42 0 1.22 0 0.01 0 1.11 0 0.00 0 168.31 0 0.38 0 53723 0 888301 0 150765 0 11102 0 2956 0 0 0 241396 0 8 0 0 0 154 0 15688 0 370 0 16220 0 54.27 0 482082 0 5897 18187 3.084110564694 888301.0 632847.0 53723.0 150765.0 11102.0 2956.0 0.0 241396.0 482082.0 71.2 6.0 17.0 1.2 0.3 0.0 27.2 54.3 43 43 43.00 38 38196943 27.1 21.5 21.6 29.8 0.0 35.8 24.0 smartseq 1069420 SRR2088516 SRP060416 SRS979822 SRX1082485 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810951: T86_P2_C11_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810951 GSM1810951 T86_P2_C11_ILC2 185307468 4309476 2016-01-28 01:00:06 197198607 185307468 4309476 1 4309476 index:0,count:4309476,average:43,stdev:0 GSM1810951_r1 6.26 7.92 0.23 121375700 153570508 92477321 121618733 126.52 131.51 0 0 0 0 0 0 55.67 74.63 5075013 1667318 5075013 1667318 62.85 70.89 5075013 1882261 5075013 1583832 20312326 16.74 6.40 0 17.65 0 1.26 0 0.32 0 0.00 0 28.92 0 2994955 0 43 0 41.39 0 1.17 0 0.01 0 1.13 0 0.00 0 272.18 0 0.37 0 275723 0 4309476 0 760798 0 54400 0 13919 0 0 0 1246202 0 14 0 0 0 741 0 68668 0 1734 0 71157 0 51.84 0 2234157 0 8198 80556 9.826299097341 4309476.0 2994955.0 275723.0 760798.0 54400.0 13919.0 0.0 1246202.0 2234157.0 69.5 6.4 17.7 1.3 0.3 0.0 28.9 51.8 43 43 43.00 38 185307468 27.0 21.4 21.5 30.1 0.0 36.2 24.4 smartseq 1069436 SRR2088517 SRP060416 SRS979821 SRX1082486 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810952: T86_P2_C12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810952 GSM1810952 T86_P2_C12_ILC2 176029874 4093718 2016-01-28 01:00:06 188846787 176029874 4093718 1 4093718 index:0,count:4093718,average:43,stdev:0 GSM1810952_r1 5.32 7.71 0.23 117597231 152434407 90276464 121440597 129.62 134.52 0 0 0 0 0 0 58.53 77.7 4812279 1690504 4812279 1690504 65.6 73.28 4812279 1894756 4812279 1594303 16716446 14.21 6.16 0 17.40 0 1.26 0 0.39 0 0.00 0 27.80 0 2888287 0 43 0 41.49 0 1.14 0 0.01 0 1.13 0 0.00 0 216.73 0 0.37 0 252167 0 4093718 0 712508 0 51414 0 15788 0 0 0 1138229 0 28 0 0 0 647 0 72658 0 1644 0 74977 0 53.15 0 2175779 0 8322 86376 10.379235760634 4093718.0 2888287.0 252167.0 712508.0 51414.0 15788.0 0.0 1138229.0 2175779.0 70.6 6.2 17.4 1.3 0.4 0.0 27.8 53.1 43 43 43.00 38 176029874 26.7 21.9 22.0 29.4 0.0 36.1 24.4 smartseq 1069452 SRR2088518 SRP060416 SRS979820 SRX1082487 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810953: T86_P2_C1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810953 GSM1810953 T86_P2_C1_ILC2 200490553 4662571 2016-01-28 01:00:06 212918718 200490553 4662571 1 4662571 index:0,count:4662571,average:43,stdev:0 GSM1810953_r1 4.42 8.99 0.27 115825437 143505223 82748796 107113560 123.9 129.44 0 0 0 0 0 0 52.52 75.44 5312992 1513963 5312992 1513963 61.79 71.7 5312992 1781065 5312992 1438967 19617420 16.94 8.16 0 18.78 0 1.28 0 0.23 0 0.00 0 36.67 0 2882488 0 43 0 41.23 0 1.16 0 0.01 0 1.13 0 0.01 0 299.74 0 0.38 0 380579 0 4662571 0 875611 0 59500 0 10882 0 0 0 1709701 0 29 0 0 0 536 0 63659 0 1856 0 66080 0 43.04 0 2006877 0 7167 79314 11.066555043951 4662571.0 2882488.0 380579.0 875611.0 59500.0 10882.0 0.0 1709701.0 2006877.0 61.8 8.2 18.8 1.3 0.2 0.0 36.7 43.0 43 43 43.00 38 200490553 26.4 21.4 21.6 30.6 0.0 36.2 24.2 smartseq 1069468 SRR2088519 SRP060416 SRS979819 SRX1082488 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810954: T86_P2_C2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810954 GSM1810954 T86_P2_C2_ILC2 76973311 1790077 2016-01-28 01:00:06 85347451 76973311 1790077 1 1790077 index:0,count:1790077,average:43,stdev:0 GSM1810954_r1 6.38 7.66 0.21 53841099 70345658 41920576 56651938 130.65 135.14 0 0 0 0 0 0 61.31 80.12 2126594 810146 2126594 810146 68.56 75.95 2126594 905825 2126594 767897 7094527 13.18 5.48 0 17.33 0 1.04 0 0.33 0 0.00 0 24.81 0 1321298 0 43 0 41.46 0 1.18 0 0.01 0 1.14 0 0.01 0 268.51 0 0.41 0 98183 0 1790077 0 310177 0 18702 0 5963 0 0 0 444114 0 19 0 0 0 303 0 34536 0 679 0 35537 0 56.48 0 1011121 0 7696 40118 5.212837837838 1790077.0 1321298.0 98183.0 310177.0 18702.0 5963.0 0.0 444114.0 1011121.0 73.8 5.5 17.3 1.0 0.3 0.0 24.8 56.5 43 43 43.00 38 76973311 27.1 21.6 22.1 29.3 0.0 35.6 23.8 smartseq 1069581 SRR2088520 SRP060416 SRS979818 SRX1082489 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810955: T86_P2_C3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810955 GSM1810955 T86_P2_C3_ILC2 162776844 3785508 2016-01-28 01:00:06 173464753 162776844 3785508 1 3785508 index:0,count:3785508,average:43,stdev:0 GSM1810955_r1 4.9 8.32 0.22 111666645 145333150 87944065 118725800 130.15 135.0 0 0 0 0 0 0 60.28 77.94 4437554 1651682 4437554 1651682 67.06 74.37 4437554 1837391 4437554 1576084 15971333 14.30 5.78 0 16.39 0 1.25 0 0.37 0 0.00 0 26.00 0 2739876 0 43 0 41.50 0 1.18 0 0.01 0 1.15 0 0.01 0 302.84 0 0.35 0 218751 0 3785508 0 620603 0 47320 0 14130 0 0 0 984182 0 39 0 0 0 506 0 67131 0 1471 0 69147 0 55.98 0 2119273 0 7478 78980 10.561647499331 3785508.0 2739876.0 218751.0 620603.0 47320.0 14130.0 0.0 984182.0 2119273.0 72.4 5.8 16.4 1.3 0.4 0.0 26.0 56.0 43 43 43.00 38 162776844 26.8 21.8 21.8 29.6 0.0 36.3 24.8 smartseq 1069596 SRR2088521 SRP060416 SRS979817 SRX1082490 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810956: T86_P2_C5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810956 GSM1810956 T86_P2_C5_ILC2 84065989 1955023 2016-01-28 01:00:06 91211680 84065989 1955023 1 1955023 index:0,count:1955023,average:43,stdev:0 GSM1810956_r1 5.74 8.53 0.25 53321725 66401824 40326928 52245897 124.53 129.56 0 0 0 0 0 0 53.97 73.0 2257714 714647 2257714 714647 60.97 69.59 2257714 807386 2257714 681278 9546409 17.90 6.79 0 17.65 0 1.24 0 0.43 0 0.00 0 30.60 0 1324152 0 43 0 41.19 0 1.19 0 0.01 0 1.15 0 0.00 0 219.94 0 0.39 0 132815 0 1955023 0 345122 0 24221 0 8500 0 0 0 598150 0 11 0 0 0 218 0 24523 0 826 0 25578 0 50.08 0 979030 0 4765 29334 6.156138509969 1955023.0 1324152.0 132815.0 345122.0 24221.0 8500.0 0.0 598150.0 979030.0 67.7 6.8 17.7 1.2 0.4 0.0 30.6 50.1 43 43 43.00 38 84065989 27.2 21.2 21.2 30.4 0.0 36.0 24.1 smartseq 1069611 SRR2088522 SRP060416 SRS979816 SRX1082491 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810957: T86_P2_C6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810957 GSM1810957 T86_P2_C6_ILC2 160815614 3739898 2016-01-28 01:00:06 172394814 160815614 3739898 1 3739898 index:0,count:3739898,average:43,stdev:0 GSM1810957_r1 2.78 11.76 0.27 84915391 100396601 61255542 77004842 118.23 125.71 0 0 0 0 0 0 50.84 72.97 3980389 1095951 3980389 1095951 56.24 70.17 3980389 1212517 3980389 1053922 17786346 20.95 9.15 0 17.48 0 1.34 0 0.24 0 0.00 0 40.77 0 2155882 0 43 0 40.78 0 1.21 0 0.01 0 1.11 0 0.00 0 203.99 0 0.41 0 342362 0 3739898 0 653860 0 50215 0 8873 0 0 0 1524928 0 15 0 0 0 345 0 43951 0 1647 0 45958 0 40.16 0 1502022 0 5585 49991 8.950940017905 3739898.0 2155882.0 342362.0 653860.0 50215.0 8873.0 0.0 1524928.0 1502022.0 57.6 9.2 17.5 1.3 0.2 0.0 40.8 40.2 43 43 43.00 38 160815614 26.5 20.8 20.9 31.8 0.0 35.9 23.6 smartseq 1069627 SRR2088523 SRP060416 SRS979815 SRX1082492 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810958: T86_P2_C7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810958 GSM1810958 T86_P2_C7_ILC2 115651295 2689565 2016-01-28 01:00:06 124272040 115651295 2689565 1 2689565 index:0,count:2689565,average:43,stdev:0 GSM1810958_r1 4.17 8.58 0.26 72894429 90851145 57099726 73957445 124.63 129.52 0 0 0 0 0 0 59.03 77.04 2959225 1065651 2959225 1065651 64.38 73.4 2959225 1162260 2959225 1015319 11477559 15.75 6.91 0 15.69 0 1.27 0 0.36 0 0.00 0 31.25 0 1805344 0 43 0 41.28 0 1.17 0 0.01 0 1.12 0 0.00 0 254.80 0 0.37 0 185950 0 2689565 0 422077 0 34186 0 9619 0 0 0 840416 0 16 0 0 0 331 0 43128 0 1029 0 44504 0 51.43 0 1383267 0 6091 49086 8.058775242161 2689565.0 1805344.0 185950.0 422077.0 34186.0 9619.0 0.0 840416.0 1383267.0 67.1 6.9 15.7 1.3 0.4 0.0 31.2 51.4 43 43 43.00 38 115651295 26.8 21.6 21.5 30.0 0.0 36.2 24.3 smartseq 1069643 SRR2088524 SRP060416 SRS979814 SRX1082493 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810959: T86_P2_C8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810959 GSM1810959 T86_P2_C8_ILC2 66009644 1535108 2016-01-28 01:00:06 71447014 66009644 1535108 1 1535108 index:0,count:1535108,average:43,stdev:0 GSM1810959_r1 6.14 7.26 0.25 45885728 59330695 35004240 46829796 129.3 133.78 0 0 0 0 0 0 57.81 77.05 1886451 651245 1886451 651245 66.38 73.19 1886451 747812 1886451 618562 6498374 14.16 5.56 0 18.33 0 1.22 0 0.35 0 0.00 0 25.04 0 1126581 0 43 0 41.42 0 1.27 0 0.01 0 1.12 0 0.01 0 345.40 0 0.36 0 85305 0 1535108 0 281407 0 18697 0 5438 0 0 0 384392 0 4 0 0 0 267 0 26002 0 674 0 26947 0 55.06 0 845174 0 6625 31260 4.718490566038 1535108.0 1126581.0 85305.0 281407.0 18697.0 5438.0 0.0 384392.0 845174.0 73.4 5.6 18.3 1.2 0.4 0.0 25.0 55.1 43 43 43.00 38 66009644 26.9 21.7 21.7 29.7 0.0 36.2 24.5 smartseq 1069659 SRR2088525 SRP060416 SRS979813 SRX1082494 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810960: T86_P2_C9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810960 GSM1810960 T86_P2_C9_ILC2 30943703 719621 2016-01-28 01:00:06 33550797 30943703 719621 1 719621 index:0,count:719621,average:43,stdev:0 GSM1810960_r1 4.18 8.55 0.19 19893843 24834089 15341828 19920973 124.83 129.85 0 0 0 0 0 0 53.94 71.44 817351 264803 817351 264803 60.55 68.01 817351 297230 817351 252082 3277040 16.47 6.70 0 16.71 0 1.36 0 0.39 0 0.00 0 30.03 0 490895 0 43 0 41.39 0 1.20 0 0.01 0 1.14 0 0.00 0 172.71 0 0.36 0 48181 0 719621 0 120248 0 9757 0 2835 0 0 0 216134 0 17 0 0 0 93 0 12507 0 275 0 12892 0 51.51 0 370647 0 5467 13872 2.537406255716 719621.0 490895.0 48181.0 120248.0 9757.0 2835.0 0.0 216134.0 370647.0 68.2 6.7 16.7 1.4 0.4 0.0 30.0 51.5 43 43 43.00 38 30943703 26.7 21.6 21.5 30.2 0.0 36.3 24.4 smartseq 1069675 SRR2088526 SRP060416 SRS979812 SRX1082495 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810961: T86_P2_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810961 GSM1810961 T86_P2_D11_NK 95211546 2214222 2016-01-28 01:00:06 102273859 95211546 2214222 1 2214222 index:0,count:2214222,average:43,stdev:0 GSM1810961_r1 4.56 7.81 0.22 63005544 81814249 50036142 67361649 129.85 134.63 0 0 0 0 0 0 59.26 75.92 2448848 915351 2448848 915351 65.29 72.25 2448848 1008514 2448848 871128 9014283 14.31 6.41 0 15.31 0 1.15 0 0.37 0 0.00 0 28.71 0 1544741 0 43 0 41.50 0 1.25 0 0.01 0 1.13 0 0.00 0 249.10 0 0.36 0 142012 0 2214222 0 339049 0 25402 0 8275 0 0 0 635804 0 39 0 0 0 400 0 40400 0 892 0 41731 0 54.45 0 1205692 0 8686 47124 5.425282063090 2214222.0 1544741.0 142012.0 339049.0 25402.0 8275.0 0.0 635804.0 1205692.0 69.8 6.4 15.3 1.1 0.4 0.0 28.7 54.5 43 43 43.00 38 95211546 26.8 22.0 22.0 29.3 0.0 36.4 24.8 smartseq 1069690 SRR2088527 SRP060416 SRS979811 SRX1082496 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810962: T86_P2_D12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810962 GSM1810962 T86_P2_D12_ILC2 134167869 3120183 2016-01-28 01:00:06 143584253 134167869 3120183 1 3120183 index:0,count:3120183,average:43,stdev:0 GSM1810962_r1 5.06 7.33 0.22 89531512 118366053 69451053 95080829 132.21 136.9 0 0 0 0 0 0 63.5 83.15 3622715 1390872 3622715 1390872 70.79 78.52 3622715 1550572 3622715 1313430 9635252 10.76 6.34 0 16.59 0 1.04 0 0.26 0 0.00 0 28.50 0 2190264 0 43 0 41.52 0 1.14 0 0.01 0 1.15 0 0.01 0 244.19 0 0.36 0 197923 0 3120183 0 517613 0 32556 0 8252 0 0 0 889111 0 28 0 0 0 570 0 66433 0 1293 0 68324 0 53.61 0 1672651 0 9668 79410 8.213694662805 3120183.0 2190264.0 197923.0 517613.0 32556.0 8252.0 0.0 889111.0 1672651.0 70.2 6.3 16.6 1.0 0.3 0.0 28.5 53.6 43 43 43.00 38 134167869 26.6 22.3 22.3 28.8 0.0 36.4 24.9 smartseq 1069705 SRR2088528 SRP060416 SRS979810 SRX1082497 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810963: T86_P2_D1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810963 GSM1810963 T86_P2_D1_ILC2 132660719 3085133 2016-01-28 01:00:06 142060994 132660719 3085133 1 3085133 index:0,count:3085133,average:43,stdev:0 GSM1810963_r1 6.4 8.13 0.25 78926408 103412720 60021326 82011943 131.02 136.64 0 0 0 0 0 0 60.55 81.22 3246927 1177178 3246927 1177178 68.31 77.22 3246927 1327980 3246927 1119095 10501640 13.31 8.09 0 16.04 0 0.98 0 0.29 0 0.00 0 35.72 0 1944041 0 43 0 41.41 0 1.19 0 0.01 0 1.11 0 0.00 0 222.13 0 0.37 0 249513 0 3085133 0 494732 0 30109 0 8899 0 0 0 1102084 0 12 0 0 0 395 0 53193 0 1268 0 54868 0 46.98 0 1449309 0 7802 63488 8.137400666496 3085133.0 1944041.0 249513.0 494732.0 30109.0 8899.0 0.0 1102084.0 1449309.0 63.0 8.1 16.0 1.0 0.3 0.0 35.7 47.0 43 43 43.00 38 132660719 26.6 21.9 21.9 29.5 0.0 36.3 24.7 smartseq 1069722 SRR2088529 SRP060416 SRS979809 SRX1082498 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810964: T86_P2_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810964 GSM1810964 T86_P2_D2_NK 97044034 2256838 2016-01-28 01:00:06 104445777 97044034 2256838 1 2256838 index:0,count:2256838,average:43,stdev:0 GSM1810964_r1 4.67 6.42 0.2 70895543 94610331 57860996 79124149 133.45 136.75 0 0 0 0 0 0 65.02 80.57 2554594 1118477 2554594 1118477 70.8 75.86 2554594 1218055 2554594 1053057 7432098 10.48 5.02 0 14.72 0 1.06 0 0.44 0 0.00 0 22.27 0 1720327 0 43 0 41.68 0 1.32 0 0.01 0 1.13 0 0.00 0 290.16 0 0.36 0 113240 0 2256838 0 332150 0 23927 0 9889 0 0 0 502695 0 19 0 0 0 403 0 55826 0 843 0 57091 0 61.51 0 1388177 0 11110 66823 6.014671467147 2256838.0 1720327.0 113240.0 332150.0 23927.0 9889.0 0.0 502695.0 1388177.0 76.2 5.0 14.7 1.1 0.4 0.0 22.3 61.5 43 43 43.00 38 97044034 26.4 22.6 22.6 28.4 0.0 36.4 25.2 smartseq 1069834 SRR2088530 SRP060416 SRS979808 SRX1082499 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810965: T86_P2_D3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810965 GSM1810965 T86_P2_D3_ILC2 150391812 3497484 2016-01-28 01:00:06 160211653 150391812 3497484 1 3497484 index:0,count:3497484,average:43,stdev:0 GSM1810965_r1 5.15 6.77 0.25 99418708 128993291 78066505 103982294 129.75 133.2 0 0 0 0 0 0 61.41 79.4 3901249 1489104 3901249 1489104 69.21 75.87 3901249 1678328 3901249 1422850 12131045 12.20 6.46 0 15.71 0 1.09 0 0.44 0 0.00 0 29.14 0 2424840 0 43 0 41.63 0 1.19 0 0.01 0 1.14 0 0.01 0 286.16 0 0.35 0 226104 0 3497484 0 549474 0 37986 0 15486 0 0 0 1019172 0 20 0 0 0 473 0 69251 0 1197 0 70941 0 53.62 0 1875366 0 8028 82390 10.262830094669 3497484.0 2424840.0 226104.0 549474.0 37986.0 15486.0 0.0 1019172.0 1875366.0 69.3 6.5 15.7 1.1 0.4 0.0 29.1 53.6 43 43 43.00 38 150391812 26.9 22.1 22.2 28.9 0.0 36.5 25.2 smartseq 1069850 SRR2088531 SRP060416 SRS979807 SRX1082500 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810966: T86_P2_D5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810966 GSM1810966 T86_P2_D5_ILC2 36158700 840900 2016-01-28 01:00:06 39221179 36158700 840900 1 840900 index:0,count:840900,average:43,stdev:0 GSM1810966_r1 3.53 8.61 0.21 22588546 28684921 17388717 22966292 126.99 132.08 0 0 0 0 0 0 58.65 77.74 924628 327691 924628 327691 65.55 74.01 924628 366237 924628 311987 3262120 14.44 7.15 0 16.31 0 1.15 0 0.35 0 0.00 0 32.05 0 558710 0 43 0 41.25 0 1.25 0 0.01 0 1.12 0 0.01 0 159.33 0 0.38 0 60131 0 840900 0 137182 0 9697 0 2972 0 0 0 269521 0 9 0 0 0 113 0 14337 0 430 0 14889 0 50.13 0 421528 0 5336 16698 3.129310344828 840900.0 558710.0 60131.0 137182.0 9697.0 2972.0 0.0 269521.0 421528.0 66.4 7.2 16.3 1.2 0.4 0.0 32.1 50.1 43 43 43.00 38 36158700 26.8 21.8 21.6 29.9 0.0 36.3 24.5 smartseq 1069866 SRR2088532 SRP060416 SRS979806 SRX1082501 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810967: T86_P2_D6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810967 GSM1810967 T86_P2_D6_ILC2 61301488 1425616 2016-01-28 01:00:06 65997126 61301488 1425616 1 1425616 index:0,count:1425616,average:43,stdev:0 GSM1810967_r1 4.33 8.2 0.23 40666760 51850645 32551046 42899629 127.5 131.79 0 0 0 0 0 0 59.45 75.59 1560481 593545 1560481 593545 64.66 71.79 1560481 645563 1560481 563679 6269135 15.42 6.39 0 14.95 0 1.11 0 0.43 0 0.00 0 28.43 0 998381 0 43 0 41.45 0 1.22 0 0.01 0 1.14 0 0.01 0 320.76 0 0.34 0 91167 0 1425616 0 213167 0 15773 0 6124 0 0 0 405338 0 8 0 0 0 181 0 25580 0 605 0 26374 0 55.08 0 785214 0 6601 28881 4.375246174822 1425616.0 998381.0 91167.0 213167.0 15773.0 6124.0 0.0 405338.0 785214.0 70.0 6.4 15.0 1.1 0.4 0.0 28.4 55.1 43 43 43.00 38 61301488 27.1 21.6 21.5 29.9 0.0 36.4 24.9 smartseq 1069881 SRR2088533 SRP060416 SRS979782 SRX1082502 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810968: T86_P2_D7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810968 GSM1810968 T86_P2_D7_ILC2 57272044 1331908 2016-01-28 01:00:06 61885397 57272044 1331908 1 1331908 index:0,count:1331908,average:43,stdev:0 GSM1810968_r1 4.77 7.28 0.23 39309581 50515967 31288448 41446635 128.51 132.47 0 0 0 0 0 0 61.93 78.97 1514370 595571 1514370 595571 68.08 74.73 1514370 654658 1514370 563615 4981296 12.67 5.88 0 15.58 0 1.05 0 0.44 0 0.00 0 26.31 0 961658 0 43 0 41.49 0 1.23 0 0.01 0 1.17 0 0.01 0 299.68 0 0.34 0 78290 0 1331908 0 207454 0 13994 0 5803 0 0 0 350453 0 10 0 0 0 239 0 27117 0 639 0 28005 0 56.63 0 754204 0 7479 30965 4.140259392967 1331908.0 961658.0 78290.0 207454.0 13994.0 5803.0 0.0 350453.0 754204.0 72.2 5.9 15.6 1.1 0.4 0.0 26.3 56.6 43 43 43.00 38 57272044 26.6 22.1 22.0 29.3 0.0 36.4 24.9 smartseq 1069897 SRR2088534 SRP060416 SRS979805 SRX1082503 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810969: T86_P2_D8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810969 GSM1810969 T86_P2_D8_ILC2 22374706 520342 2016-01-28 01:00:06 24380187 22374706 520342 1 520342 index:0,count:520342,average:43,stdev:0 GSM1810969_r1 5.0 9.23 0.23 12968505 16524521 9579046 12838268 127.42 134.02 0 0 0 0 0 0 56.06 77.73 568112 181160 568112 181160 63.89 74.23 568112 206464 568112 172987 1982146 15.28 8.06 0 17.32 0 1.30 0 0.24 0 0.00 0 36.35 0 323164 0 43 0 41.10 0 1.24 0 0.01 0 1.14 0 0.00 0 98.59 0 0.40 0 41964 0 520342 0 90112 0 6775 0 1233 0 0 0 189170 0 5 0 0 0 64 0 7208 0 250 0 7527 0 44.79 0 233052 0 3501 8236 2.352470722651 520342.0 323164.0 41964.0 90112.0 6775.0 1233.0 0.0 189170.0 233052.0 62.1 8.1 17.3 1.3 0.2 0.0 36.4 44.8 43 43 43.00 38 22374706 26.7 21.5 21.3 30.5 0.0 36.1 24.1 smartseq 1069912 SRR2088535 SRP060416 SRS979804 SRX1082504 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810970: T86_P2_E10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810970 GSM1810970 T86_P2_E10_ILC2 44457141 1033887 2016-01-28 01:00:06 48331721 44457141 1033887 1 1033887 index:0,count:1033887,average:43,stdev:0 GSM1810970_r1 6.94 7.2 0.19 31312269 41572002 23888137 32799755 132.77 137.31 0 0 0 0 0 0 60.58 80.65 1266182 464054 1266182 464054 69.75 76.52 1266182 534302 1266182 440293 3803095 12.15 5.38 0 18.44 0 1.18 0 0.31 0 0.00 0 24.41 0 766054 0 43 0 41.52 0 1.18 0 0.01 0 1.15 0 0.01 0 206.78 0 0.36 0 55674 0 1033887 0 190679 0 12237 0 3248 0 0 0 252348 0 14 0 0 0 131 0 20883 0 409 0 21437 0 55.65 0 575375 0 7369 25023 3.395711765504 1033887.0 766054.0 55674.0 190679.0 12237.0 3248.0 0.0 252348.0 575375.0 74.1 5.4 18.4 1.2 0.3 0.0 24.4 55.7 43 43 43.00 38 44457141 26.6 22.0 21.9 29.4 0.0 36.2 24.7 smartseq 1069928 SRR2088536 SRP060416 SRS979803 SRX1082505 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810971: T86_P2_E11_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810971 GSM1810971 T86_P2_E11_ILC2 196515590 4570130 2016-01-28 01:00:06 209305255 196515590 4570130 1 4570130 index:0,count:4570130,average:43,stdev:0 GSM1810971_r1 6.66 7.72 0.23 135128142 179817171 101615714 140281658 133.07 138.05 0 0 0 0 0 0 59.74 80.83 5634373 1979854 5634373 1979854 69.05 76.72 5634373 2288477 5634373 1879374 15847428 11.73 5.76 0 18.92 0 1.13 0 0.31 0 0.00 0 26.05 0 3314119 0 43 0 41.48 0 1.15 0 0.01 0 1.15 0 0.00 0 293.79 0 0.38 0 263185 0 4570130 0 864568 0 51555 0 13992 0 0 0 1190464 0 12 0 0 0 584 0 88115 0 1831 0 90542 0 53.60 0 2449551 0 10176 105797 10.396717767296 4570130.0 3314119.0 263185.0 864568.0 51555.0 13992.0 0.0 1190464.0 2449551.0 72.5 5.8 18.9 1.1 0.3 0.0 26.0 53.6 43 43 43.00 38 196515590 26.5 22.0 22.0 29.4 0.0 36.2 24.6 smartseq 1069944 SRR2088537 SRP060416 SRS979802 SRX1082506 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810972: T86_P2_E12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810972 GSM1810972 T86_P2_E12_ILC2 227890454 5299778 2016-01-28 01:00:06 241562026 227890454 5299778 1 5299778 index:0,count:5299778,average:43,stdev:0 GSM1810972_r1 6.51 7.39 0.19 157917159 207212524 120932852 164168232 131.22 135.75 0 0 0 0 0 0 61.04 81.04 6463975 2358473 6463975 2358473 69.31 76.78 6463975 2678091 6463975 2234496 19835198 12.56 5.67 0 17.99 0 1.12 0 0.31 0 0.00 0 25.67 0 3863775 0 43 0 41.55 0 1.17 0 0.01 0 1.11 0 0.00 0 293.53 0 0.36 0 300246 0 5299778 0 953539 0 59230 0 16469 0 0 0 1360304 0 11 0 0 0 816 0 105532 0 2106 0 108465 0 54.91 0 2910236 0 9674 124715 12.891771759355 5299778.0 3863775.0 300246.0 953539.0 59230.0 16469.0 0.0 1360304.0 2910236.0 72.9 5.7 18.0 1.1 0.3 0.0 25.7 54.9 43 43 43.00 38 227890454 26.5 22.1 22.2 29.2 0.0 36.3 24.7 smartseq 1069960 SRR2088538 SRP060416 SRS979801 SRX1082507 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810973: T86_P2_E1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810973 GSM1810973 T86_P2_E1_ILC2 183310118 4263026 2016-01-28 01:00:06 195725364 183310118 4263026 1 4263026 index:0,count:4263026,average:43,stdev:0 GSM1810973_r1 3.33 9.01 0.29 107903680 133383454 79483835 102529001 123.61 128.99 0 0 0 0 0 0 54.75 76.19 4761913 1468052 4761913 1468052 62.44 72.39 4761913 1674264 4761913 1394879 17515924 16.23 7.82 0 17.70 0 1.34 0 0.35 0 0.00 0 35.41 0 2681409 0 43 0 41.25 0 1.20 0 0.01 0 1.14 0 0.00 0 269.24 0 0.39 0 333402 0 4263026 0 754546 0 57178 0 14981 0 0 0 1509458 0 35 0 0 0 558 0 65923 0 1801 0 68317 0 45.20 0 1926863 0 7740 80766 10.434883720930 4263026.0 2681409.0 333402.0 754546.0 57178.0 14981.0 0.0 1509458.0 1926863.0 62.9 7.8 17.7 1.3 0.4 0.0 35.4 45.2 43 43 43.00 38 183310118 26.4 21.7 21.8 30.0 0.0 36.1 24.2 smartseq 1069976 SRR2088539 SRP060416 SRS979800 SRX1082508 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810974: T86_P2_E2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810974 GSM1810974 T86_P2_E2_ILC2 81086003 1885721 2016-01-28 01:00:06 89168099 81086003 1885721 1 1885721 index:0,count:1885721,average:43,stdev:0 GSM1810974_r1 5.71 7.84 0.24 54214938 70908626 41850294 56810665 130.79 135.75 0 0 0 0 0 0 62.57 82.61 2195561 835263 2195561 835263 69.81 78.55 2195561 931849 2195561 794198 6546607 12.08 6.12 0 17.17 0 1.15 0 0.27 0 0.00 0 27.79 0 1334903 0 43 0 41.39 0 1.16 0 0.01 0 1.12 0 0.01 0 199.66 0 0.40 0 115417 0 1885721 0 323826 0 21760 0 5050 0 0 0 524008 0 6 0 0 0 195 0 35232 0 738 0 36171 0 53.62 0 1011077 0 7035 40953 5.821321961620 1885721.0 1334903.0 115417.0 323826.0 21760.0 5050.0 0.0 524008.0 1011077.0 70.8 6.1 17.2 1.2 0.3 0.0 27.8 53.6 43 43 43.00 38 81086003 26.9 21.8 22.0 29.3 0.0 35.8 23.9 smartseq 1070088 SRR2088540 SRP060416 SRS979799 SRX1082509 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810975: T86_P2_E3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810975 GSM1810975 T86_P2_E3_ILC2 190644757 4433599 2016-01-28 01:00:06 202803856 190644757 4433599 1 4433599 index:0,count:4433599,average:43,stdev:0 GSM1810975_r1 5.54 6.41 0.21 144127467 191616295 113286318 154161097 132.95 136.08 0 0 0 0 0 0 63.4 81.6 5527345 2217835 5527345 2217835 72.15 77.08 5527345 2524049 5527345 2094839 15693767 10.89 4.37 0 17.61 0 1.02 0 0.42 0 0.00 0 19.66 0 3498395 0 43 0 41.68 0 1.23 0 0.01 0 1.16 0 0.01 0 285.02 0 0.34 0 193820 0 4433599 0 780553 0 45286 0 18416 0 0 0 871502 0 26 0 0 0 797 0 104649 0 1558 0 107030 0 61.30 0 2717842 0 12576 127935 10.172948473282 4433599.0 3498395.0 193820.0 780553.0 45286.0 18416.0 0.0 871502.0 2717842.0 78.9 4.4 17.6 1.0 0.4 0.0 19.7 61.3 43 43 43.00 38 190644757 26.5 22.4 22.4 28.6 0.0 36.4 25.2 smartseq 1070106 SRR2088541 SRP060416 SRS979798 SRX1082510 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810976: T86_P2_E5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810976 GSM1810976 T86_P2_E5_ILC2 123793259 2878913 2016-01-28 01:00:06 133171080 123793259 2878913 1 2878913 index:0,count:2878913,average:43,stdev:0 GSM1810976_r1 5.82 8.07 0.22 85261067 110892327 65272342 87741762 130.06 134.42 0 0 0 0 0 0 60.65 80.65 3462952 1271361 3462952 1271361 68.82 76.74 3462952 1442688 3462952 1209715 10848505 12.72 5.70 0 18.06 0 1.12 0 0.28 0 0.00 0 25.79 0 2096257 0 43 0 41.41 0 1.18 0 0.01 0 1.14 0 0.00 0 280.11 0 0.37 0 164186 0 2878913 0 519880 0 32222 0 8039 0 0 0 742395 0 38 0 0 0 370 0 55274 0 1298 0 56980 0 54.76 0 1576377 0 8046 65428 8.131742480736 2878913.0 2096257.0 164186.0 519880.0 32222.0 8039.0 0.0 742395.0 1576377.0 72.8 5.7 18.1 1.1 0.3 0.0 25.8 54.8 43 43 43.00 38 123793259 26.7 21.9 21.8 29.6 0.0 36.2 24.5 smartseq 1070122 SRR2088542 SRP060416 SRS979797 SRX1082511 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810979: T86_P2_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810979 GSM1810979 T86_P2_E6_NK 160493673 3732411 2016-01-28 01:00:06 171611032 160493673 3732411 1 3732411 index:0,count:3732411,average:43,stdev:0 GSM1810979_r1 4.05 7.2 0.2 115968797 148442523 92837439 122766529 128.0 132.24 0 0 0 0 0 0 59.18 74.99 4428823 1677311 4428823 1677311 64.95 71.08 4428823 1840823 4428823 1589960 15935542 13.74 4.87 0 16.00 0 1.74 0 0.48 0 0.00 0 21.84 0 2834196 0 43 0 41.50 0 1.28 0 0.01 0 1.16 0 0.01 0 263.46 0 0.35 0 181694 0 3732411 0 597361 0 64974 0 18058 0 0 0 815183 0 45 0 0 0 616 0 73878 0 1646 0 76185 0 59.93 0 2236835 0 9512 86096 9.051303616484 3732411.0 2834196.0 181694.0 597361.0 64974.0 18058.0 0.0 815183.0 2236835.0 75.9 4.9 16.0 1.7 0.5 0.0 21.8 59.9 43 43 43.00 38 160493673 26.6 22.1 22.1 29.2 0.0 36.3 24.8 smartseq 1070137 SRR2088543 SRP060416 SRS979796 SRX1082512 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810981: T86_P2_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810981 GSM1810981 T86_P2_E7_NK 115771265 2692355 2016-01-28 01:00:06 124537274 115771265 2692355 1 2692355 index:0,count:2692355,average:43,stdev:0 GSM1810981_r1 4.0 8.02 0.26 80290057 103620042 61810516 83017998 129.06 134.31 0 0 0 0 0 0 55.16 72.98 3264710 1090661 3264710 1090661 61.89 68.77 3264710 1223773 3264710 1027745 12897384 16.06 5.50 0 17.93 0 1.13 0 0.44 0 0.00 0 24.99 0 1977235 0 43 0 41.36 0 1.24 0 0.01 0 1.13 0 0.01 0 372.79 0 0.36 0 147971 0 2692355 0 482847 0 30550 0 11875 0 0 0 672695 0 17 0 0 0 374 0 50905 0 1270 0 52566 0 55.50 0 1494388 0 6146 60408 9.828831760495 2692355.0 1977235.0 147971.0 482847.0 30550.0 11875.0 0.0 672695.0 1494388.0 73.4 5.5 17.9 1.1 0.4 0.0 25.0 55.5 43 43 43.00 38 115771265 26.8 21.7 21.6 29.9 0.0 36.2 24.5 smartseq 1070153 SRR2088544 SRP060416 SRS979795 SRX1082513 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810983: T86_P2_E8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810983 GSM1810983 T86_P2_E8_ILC2 70958385 1650195 2016-01-28 01:00:06 76898769 70958385 1650195 1 1650195 index:0,count:1650195,average:43,stdev:0 GSM1810983_r1 5.73 7.57 0.22 49180413 64128279 38238924 51544568 130.39 134.8 0 0 0 0 0 0 59.87 78.37 1960882 723315 1960882 723315 67.67 74.74 1960882 817498 1960882 689818 6271928 12.75 5.62 0 17.28 0 1.18 0 0.31 0 0.00 0 25.29 0 1208148 0 43 0 41.43 0 1.22 0 0.01 0 1.14 0 0.01 0 185.65 0 0.37 0 92739 0 1650195 0 285151 0 19476 0 5180 0 0 0 417391 0 14 0 0 0 234 0 30717 0 715 0 31680 0 55.93 0 922997 0 7422 36293 4.889921853948 1650195.0 1208148.0 92739.0 285151.0 19476.0 5180.0 0.0 417391.0 922997.0 73.2 5.6 17.3 1.2 0.3 0.0 25.3 55.9 43 43 43.00 38 70958385 26.9 21.8 21.7 29.7 0.0 36.2 24.4 smartseq 1070169 SRR2088545 SRP060416 SRS979794 SRX1082514 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810985: T86_P2_F10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810985 GSM1810985 T86_P2_F10_ILC2 44873295 1043565 2016-01-28 01:00:06 48806977 44873295 1043565 1 1043565 index:0,count:1043565,average:43,stdev:0 GSM1810985_r1 6.97 7.3 0.2 31109294 41007612 23472056 32051765 131.82 136.55 0 0 0 0 0 0 60.15 81.02 1291399 457639 1291399 457639 69.78 76.85 1291399 530942 1291399 434132 3850301 12.38 5.70 0 18.78 0 1.19 0 0.35 0 0.00 0 25.55 0 760855 0 43 0 41.55 0 1.16 0 0.01 0 1.10 0 0.01 0 208.71 0 0.37 0 59459 0 1043565 0 195976 0 12369 0 3679 0 0 0 266662 0 8 0 0 0 165 0 21234 0 418 0 21825 0 54.13 0 564879 0 7250 24905 3.435172413793 1043565.0 760855.0 59459.0 195976.0 12369.0 3679.0 0.0 266662.0 564879.0 72.9 5.7 18.8 1.2 0.4 0.0 25.6 54.1 43 43 43.00 38 44873295 26.6 22.0 22.0 29.5 0.0 36.2 24.6 smartseq 1070186 SRR2088546 SRP060416 SRS979793 SRX1082515 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810987: T86_P2_F11_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810987 GSM1810987 T86_P2_F11_ILC2 158208223 3679261 2016-01-28 01:00:06 169189040 158208223 3679261 1 3679261 index:0,count:3679261,average:43,stdev:0 GSM1810987_r1 4.49 9.53 0.24 90111616 108057137 64727804 81361944 119.91 125.7 0 0 0 0 0 0 51.38 73.59 4157085 1159674 4157085 1159674 59.14 70.3 4157085 1334794 4157085 1107871 17047364 18.92 8.22 0 18.52 0 1.29 0 0.27 0 0.00 0 37.10 0 2257132 0 43 0 41.07 0 1.21 0 0.01 0 1.17 0 0.01 0 301.03 0 0.42 0 302386 0 3679261 0 681273 0 47345 0 9934 0 0 0 1364850 0 14 0 0 0 381 0 47906 0 1554 0 49855 0 42.83 0 1575859 0 6414 57423 8.952759588400 3679261.0 2257132.0 302386.0 681273.0 47345.0 9934.0 0.0 1364850.0 1575859.0 61.3 8.2 18.5 1.3 0.3 0.0 37.1 42.8 43 43 43.00 38 158208223 26.9 21.1 21.2 30.7 0.0 36.1 23.9 smartseq 1070201 SRR2088547 SRP060416 SRS979792 SRX1082516 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810988: T86_P2_F12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810988 GSM1810988 T86_P2_F12_ILC2 237384338 5520566 2016-01-28 01:00:06 250828175 237384338 5520566 1 5520566 index:0,count:5520566,average:43,stdev:0 GSM1810988_r1 5.16 6.68 0.21 174225026 230065286 135163558 183688023 132.05 135.9 0 0 0 0 0 0 60.87 79.5 6881144 2575407 6881144 2575407 69.92 75.28 6881144 2958178 6881144 2438733 20738219 11.90 4.85 0 17.96 0 1.09 0 0.42 0 0.00 0 21.85 0 4230911 0 43 0 41.72 0 1.18 0 0.01 0 1.13 0 0.00 0 354.89 0 0.35 0 267544 0 5520566 0 991362 0 60020 0 23338 0 0 0 1206297 0 39 0 0 0 895 0 124354 0 2026 0 127314 0 58.68 0 3239549 0 12041 148802 12.357943692384 5520566.0 4230911.0 267544.0 991362.0 60020.0 23338.0 0.0 1206297.0 3239549.0 76.6 4.8 18.0 1.1 0.4 0.0 21.9 58.7 43 43 43.00 38 237384338 26.5 22.3 22.4 28.9 0.0 36.4 25.0 smartseq 1070217 SRR2088548 SRP060416 SRS979791 SRX1082517 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810989: T86_P2_F1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810989 GSM1810989 T86_P2_F1_ILC2 215598388 5013916 2016-01-28 01:00:06 228828993 215598388 5013916 1 5013916 index:0,count:5013916,average:43,stdev:0 GSM1810989_r1 5.64 7.39 0.24 143733279 187611654 107773722 145602964 130.53 135.1 0 0 0 0 0 0 57.62 78.17 6010572 2023853 6010572 2023853 67.02 73.73 6010572 2353923 6010572 1908871 19239625 13.39 6.35 0 18.42 0 1.18 0 0.32 0 0.00 0 28.45 0 3512397 0 43 0 41.63 0 1.20 0 0.01 0 1.14 0 0.00 0 328.18 0 0.37 0 318459 0 5013916 0 923443 0 58967 0 16209 0 0 0 1426343 0 46 0 0 0 732 0 94769 0 2028 0 97575 0 51.64 0 2588954 0 9736 116744 11.990961380444 5013916.0 3512397.0 318459.0 923443.0 58967.0 16209.0 0.0 1426343.0 2588954.0 70.1 6.4 18.4 1.2 0.3 0.0 28.4 51.6 43 43 43.00 38 215598388 26.2 22.1 22.3 29.4 0.0 36.3 24.6 smartseq 1070234 SRR2088549 SRP060416 SRS979790 SRX1082518 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810990: T86_P2_F2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810990 GSM1810990 T86_P2_F2_ILC2 127065215 2955005 2016-01-28 01:00:06 138990449 127065215 2955005 1 2955005 index:0,count:2955005,average:43,stdev:0 GSM1810990_r1 6.51 6.99 0.18 96096467 129351758 76983099 106174424 134.61 137.92 0 0 0 0 0 0 66.03 83.41 3587255 1539011 3587255 1539011 73.47 79.12 3587255 1712519 3587255 1459794 9381159 9.76 4.39 0 16.44 0 1.00 0 0.29 0 0.00 0 19.83 0 2330907 0 43 0 41.72 0 1.15 0 0.01 0 1.14 0 0.01 0 265.95 0 0.38 0 129825 0 2955005 0 485880 0 29543 0 8693 0 0 0 585862 0 25 0 0 0 452 0 71430 0 1082 0 72989 0 62.44 0 1845027 0 11564 83632 7.232099619509 2955005.0 2330907.0 129825.0 485880.0 29543.0 8693.0 0.0 585862.0 1845027.0 78.9 4.4 16.4 1.0 0.3 0.0 19.8 62.4 43 43 43.00 38 127065215 26.6 22.3 22.6 28.5 0.0 35.9 24.4 smartseq 1070346 SRR2088550 SRP060416 SRS979789 SRX1082519 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810991: T86_P2_F3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810991 GSM1810991 T86_P2_F3_ILC2 261429379 6079753 2016-01-28 01:00:06 275628199 261429379 6079753 1 6079753 index:0,count:6079753,average:43,stdev:0 GSM1810991_r1 11.51 5.65 0.16 209675666 290334376 169810480 240080980 138.47 141.38 0 0 0 0 0 0 60.2 75.02 7661047 3043311 7661047 3043311 67.5 71.55 7661047 3412328 7661047 2902835 29152050 13.90 3.36 0 16.42 0 1.11 0 0.82 0 0.00 0 14.92 0 5054993 0 43 0 41.86 0 1.12 0 0.01 0 1.10 0 0.01 0 336.72 0 0.34 0 204106 0 6079753 0 998109 0 67643 0 49993 0 0 0 907124 0 32 0 0 0 921 0 104093 0 2315 0 107361 0 66.73 0 4056884 0 10320 120824 11.707751937984 6079753.0 5054993.0 204106.0 998109.0 67643.0 49993.0 0.0 907124.0 4056884.0 83.1 3.4 16.4 1.1 0.8 0.0 14.9 66.7 43 43 43.00 38 261429379 26.8 22.3 22.4 28.6 0.0 36.5 25.5 smartseq 1070361 SRR2088551 SRP060416 SRS979788 SRX1082520 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810992: T86_P2_F5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810992 GSM1810992 T86_P2_F5_ILC2 136553122 3175654 2016-01-28 01:00:06 146563903 136553122 3175654 1 3175654 index:0,count:3175654,average:43,stdev:0 GSM1810992_r1 3.31 7.67 0.2 93555547 118693771 75222467 98869992 126.87 131.44 0 0 0 0 0 0 60.68 76.83 3589814 1395751 3589814 1395751 65.6 73.03 3589814 1508819 3589814 1326670 13288410 14.20 5.78 0 15.23 0 1.07 0 0.38 0 0.00 0 26.12 0 2300188 0 43 0 41.41 0 1.23 0 0.01 0 1.17 0 0.01 0 259.83 0 0.37 0 183482 0 3175654 0 483617 0 33991 0 12034 0 0 0 829441 0 33 0 0 0 494 0 63719 0 1440 0 65686 0 57.20 0 1816571 0 9243 73086 7.907172995781 3175654.0 2300188.0 183482.0 483617.0 33991.0 12034.0 0.0 829441.0 1816571.0 72.4 5.8 15.2 1.1 0.4 0.0 26.1 57.2 43 43 43.00 38 136553122 26.8 21.9 21.9 29.5 0.0 36.2 24.5 smartseq 1070377 SRR2088552 SRP060416 SRS979787 SRX1082521 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810993: T86_P2_F6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810993 GSM1810993 T86_P2_F6_ILC2 175342390 4077730 2016-01-28 01:00:06 187584512 175342390 4077730 1 4077730 index:0,count:4077730,average:43,stdev:0 GSM1810993_r1 5.01 8.54 0.26 112774775 144443796 87292265 116401489 128.08 133.35 0 0 0 0 0 0 59.62 78.68 4597335 1661670 4597335 1661670 65.76 74.63 4597335 1832836 4597335 1576087 16650842 14.76 6.71 0 16.56 0 1.17 0 0.29 0 0.00 0 30.19 0 2787099 0 43 0 41.33 0 1.19 0 0.01 0 1.15 0 0.01 0 299.59 0 0.37 0 273448 0 4077730 0 675153 0 47675 0 11714 0 0 0 1231242 0 13 0 0 0 481 0 69072 0 1670 0 71236 0 51.79 0 2111946 0 8114 79450 9.791718018240 4077730.0 2787099.0 273448.0 675153.0 47675.0 11714.0 0.0 1231242.0 2111946.0 68.3 6.7 16.6 1.2 0.3 0.0 30.2 51.8 43 43 43.00 38 175342390 26.8 21.6 21.6 30.0 0.0 36.1 24.3 smartseq 1070393 SRR2088553 SRP060416 SRS979786 SRX1082522 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810994: T86_P2_F7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810994 GSM1810994 T86_P2_F7_ILC2 137791350 3204450 2016-01-28 01:00:06 147760723 137791350 3204450 1 3204450 index:0,count:3204450,average:43,stdev:0 GSM1810994_r1 6.13 6.76 0.24 102379003 137230339 80570312 111163113 134.04 137.97 0 0 0 0 0 0 64.76 83.34 3967676 1615506 3967676 1615506 72.91 79.06 3967676 1818802 3967676 1532423 10364179 10.12 4.61 0 17.36 0 1.04 0 0.33 0 0.00 0 20.78 0 2494682 0 43 0 41.56 0 1.15 0 0.01 0 1.12 0 0.00 0 240.33 0 0.36 0 147580 0 3204450 0 556259 0 33391 0 10553 0 0 0 665824 0 40 0 0 0 471 0 73501 0 1359 0 75371 0 60.49 0 1938423 0 11278 88641 7.859638233729 3204450.0 2494682.0 147580.0 556259.0 33391.0 10553.0 0.0 665824.0 1938423.0 77.9 4.6 17.4 1.0 0.3 0.0 20.8 60.5 43 43 43.00 38 137791350 26.6 22.3 22.2 28.9 0.0 36.3 24.9 smartseq 1070409 SRR2088554 SRP060416 SRS979785 SRX1082523 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810995: T86_P2_F8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810995 GSM1810995 T86_P2_F8_ILC2 76660013 1782791 2016-01-28 01:00:06 83036234 76660013 1782791 1 1782791 index:0,count:1782791,average:43,stdev:0 GSM1810995_r1 5.49 7.27 0.24 52567882 68178250 40493791 54475827 129.7 134.53 0 0 0 0 0 0 60.32 79.68 2158822 778887 2158822 778887 68.29 75.85 2158822 881728 2158822 741429 6802898 12.94 5.79 0 17.59 0 1.19 0 0.39 0 0.00 0 26.00 0 1291166 0 43 0 41.42 0 1.21 0 0.01 0 1.10 0 0.01 0 267.42 0 0.38 0 103162 0 1782791 0 313625 0 21205 0 6953 0 0 0 463467 0 3 0 0 0 243 0 33916 0 739 0 34901 0 54.83 0 977541 0 7702 40048 5.199688392625 1782791.0 1291166.0 103162.0 313625.0 21205.0 6953.0 0.0 463467.0 977541.0 72.4 5.8 17.6 1.2 0.4 0.0 26.0 54.8 43 43 43.00 38 76660013 26.8 21.9 21.8 29.5 0.0 36.2 24.4 smartseq 1070425 SRR2088555 SRP060416 SRS979784 SRX1082524 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810996: T86_P2_F9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810996 GSM1810996 T86_P2_F9_ILC2 35989538 836966 2016-01-28 01:00:06 39007145 35989538 836966 1 836966 index:0,count:836966,average:43,stdev:0 GSM1810996_r1 6.02 5.15 0.15 28732470 38271363 23830032 32327903 133.2 135.66 0 0 0 0 0 0 64.67 78.68 1005489 448089 1005489 448089 70.68 74.65 1005489 489769 1005489 425161 3370939 11.73 3.48 0 14.74 0 1.06 0 0.49 0 0.00 0 15.67 0 692901 0 43 0 41.84 0 1.21 0 0.01 0 1.14 0 0.01 0 200.87 0 0.33 0 29139 0 836966 0 123398 0 8848 0 4078 0 0 0 131139 0 11 0 0 0 148 0 20315 0 343 0 20817 0 68.04 0 569503 0 9210 23794 2.583496199783 836966.0 692901.0 29139.0 123398.0 8848.0 4078.0 0.0 131139.0 569503.0 82.8 3.5 14.7 1.1 0.5 0.0 15.7 68.0 43 43 43.00 38 35989538 26.7 22.3 22.4 28.6 0.0 36.5 25.4 smartseq 1070442 SRR2088556 SRP060416 SRS979783 SRX1082525 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810997: T86_P2_G10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810997 GSM1810997 T86_P2_G10_ILC2 51398846 1195322 2016-01-28 01:00:06 55590131 51398846 1195322 1 1195322 index:0,count:1195322,average:43,stdev:0 GSM1810997_r1 4.63 6.33 0.21 39210542 52137880 31107217 42459218 132.97 136.49 0 0 0 0 0 0 66.01 84.08 1509617 626204 1509617 626204 73.75 79.09 1509617 699633 1509617 588985 3851355 9.82 4.27 0 17.05 0 1.09 0 0.41 0 0.00 0 19.15 0 948593 0 43 0 41.77 0 1.20 0 0.01 0 1.13 0 0.01 0 358.60 0 0.34 0 51011 0 1195322 0 203849 0 12973 0 4905 0 0 0 228851 0 10 0 0 0 176 0 32773 0 457 0 33416 0 62.30 0 744744 0 9049 38516 4.256381920654 1195322.0 948593.0 51011.0 203849.0 12973.0 4905.0 0.0 228851.0 744744.0 79.4 4.3 17.1 1.1 0.4 0.0 19.1 62.3 43 43 43.00 38 51398846 26.5 22.5 22.5 28.6 0.0 36.4 25.3 smartseq 1070458 SRR2088557 SRP060416 SRS980041 SRX1082526 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810998: T86_P2_G12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810998 GSM1810998 T86_P2_G12_ILC2 196725473 4575011 2016-01-28 01:00:06 208381726 196725473 4575011 1 4575011 index:0,count:4575011,average:43,stdev:0 GSM1810998_r1 7.16 7.33 0.24 129368925 169698386 94895883 129608749 131.17 136.58 0 0 0 0 0 0 57.47 79.8 5606584 1827335 5606584 1827335 67.96 75.82 5606584 2160958 5606584 1736224 17779056 13.74 6.40 0 19.45 0 1.29 0 0.33 0 0.00 0 28.87 0 3179877 0 43 0 41.44 0 1.12 0 0.01 0 1.14 0 0.00 0 249.55 0 0.38 0 293010 0 4575011 0 890051 0 58948 0 15254 0 0 0 1320932 0 27 0 0 0 593 0 79232 0 1794 0 81646 0 50.05 0 2289826 0 8804 100273 11.389482053612 4575011.0 3179877.0 293010.0 890051.0 58948.0 15254.0 0.0 1320932.0 2289826.0 69.5 6.4 19.5 1.3 0.3 0.0 28.9 50.1 43 43 43.00 38 196725473 26.6 21.9 21.9 29.6 0.0 36.3 24.6 smartseq 1070475 SRR2088558 SRP060416 SRS980044 SRX1082527 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810999: T86_P2_G1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1810999 GSM1810999 T86_P2_G1_ILC2 206762447 4808429 2016-01-28 01:00:06 219000614 206762447 4808429 1 4808429 index:0,count:4808429,average:43,stdev:0 GSM1810999_r1 7.56 7.94 0.22 139360313 187000772 102998840 143223956 134.19 139.05 0 0 0 0 0 0 60.15 82.8 5822378 2052847 5822378 2052847 70.54 78.36 5822378 2407428 5822378 1942794 16102909 11.55 6.19 0 19.41 0 1.05 0 0.19 0 0.00 0 27.78 0 3412864 0 43 0 41.54 0 1.20 0 0.01 0 1.17 0 0.00 0 274.77 0 0.37 0 297867 0 4808429 0 933507 0 50624 0 9316 0 0 0 1335625 0 43 0 0 0 628 0 98867 0 1693 0 101231 0 51.56 0 2479357 0 10107 119348 11.808449589393 4808429.0 3412864.0 297867.0 933507.0 50624.0 9316.0 0.0 1335625.0 2479357.0 71.0 6.2 19.4 1.1 0.2 0.0 27.8 51.6 43 43 43.00 38 206762447 26.2 22.1 22.2 29.6 0.0 36.3 24.6 smartseq 1070491 SRR2088559 SRP060416 SRS979877 SRX1082528 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811000: T86_P2_G3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811000 GSM1811000 T86_P2_G3_ILC2 184709811 4295577 2016-01-28 01:00:06 195505271 184709811 4295577 1 4295577 index:0,count:4295577,average:43,stdev:0 GSM1811000_r1 3.46 7.65 0.22 130090946 165803851 104629432 137407550 127.45 131.33 0 0 0 0 0 0 60.87 76.92 4974949 1934788 4974949 1934788 66.59 73.32 4974949 2116488 4974949 1844304 18159088 13.96 5.42 0 15.44 0 1.06 0 0.52 0 0.00 0 24.42 0 3178548 0 43 0 41.60 0 1.28 0 0.01 0 1.15 0 0.01 0 303.22 0 0.34 0 232672 0 4295577 0 663282 0 45560 0 22425 0 0 0 1049044 0 43 0 0 0 698 0 90775 0 1588 0 93104 0 58.55 0 2515266 0 9895 105591 10.671147043962 4295577.0 3178548.0 232672.0 663282.0 45560.0 22425.0 0.0 1049044.0 2515266.0 74.0 5.4 15.4 1.1 0.5 0.0 24.4 58.6 43 43 43.00 38 184709811 26.9 21.9 22.0 29.2 0.0 36.4 25.0 smartseq 1070603 SRR2088560 SRP060416 SRS979880 SRX1082529 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811001: T86_P2_G5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811001 GSM1811001 T86_P2_G5_ILC2 123308735 2867645 2016-01-28 01:00:06 132123261 123308735 2867645 1 2867645 index:0,count:2867645,average:43,stdev:0 GSM1811001_r1 5.39 9.28 0.26 73390301 91799296 54050281 70938151 125.08 131.24 0 0 0 0 0 0 54.34 75.72 3261667 996679 3261667 996679 62.04 72.58 3261667 1137859 3261667 955351 12524596 17.07 7.58 0 18.06 0 1.27 0 0.31 0 0.00 0 34.46 0 1834044 0 43 0 41.06 0 1.19 0 0.01 0 1.15 0 0.01 0 279.01 0 0.40 0 217378 0 2867645 0 517807 0 36333 0 9014 0 0 0 988254 0 12 0 0 0 358 0 39463 0 1265 0 41098 0 45.90 0 1316237 0 5783 46808 8.094068822411 2867645.0 1834044.0 217378.0 517807.0 36333.0 9014.0 0.0 988254.0 1316237.0 64.0 7.6 18.1 1.3 0.3 0.0 34.5 45.9 43 43 43.00 38 123308735 27.0 21.2 21.2 30.6 0.0 36.1 24.0 smartseq 1070619 SRR2088561 SRP060416 SRS980040 SRX1082530 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811002: T86_P2_G6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811002 GSM1811002 T86_P2_G6_ILC2 134394135 3125445 2016-01-28 01:00:06 144567774 134394135 3125445 1 3125445 index:0,count:3125445,average:43,stdev:0 GSM1811002_r1 5.34 8.21 0.21 89839623 114848621 69480137 91983064 127.84 132.39 0 0 0 0 0 0 58.74 77.44 3653900 1300772 3653900 1300772 65.56 73.56 3653900 1451812 3653900 1235591 13254193 14.75 6.09 0 17.11 0 1.16 0 0.37 0 0.00 0 27.62 0 2214390 0 43 0 41.37 0 1.18 0 0.01 0 1.15 0 0.01 0 304.10 0 0.37 0 190455 0 3125445 0 534733 0 36402 0 11465 0 0 0 863188 0 1 0 0 0 367 0 48194 0 1279 0 49841 0 53.74 0 1679657 0 5770 56952 9.870363951473 3125445.0 2214390.0 190455.0 534733.0 36402.0 11465.0 0.0 863188.0 1679657.0 70.9 6.1 17.1 1.2 0.4 0.0 27.6 53.7 43 43 43.00 38 134394135 26.8 21.6 21.6 29.9 0.0 36.2 24.4 smartseq 1070634 SRR2088562 SRP060416 SRS979878 SRX1082531 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811003: T86_P2_G7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811003 GSM1811003 T86_P2_G7_ILC2 146142767 3398669 2016-01-28 01:00:06 157135782 146142767 3398669 1 3398669 index:0,count:3398669,average:43,stdev:0 GSM1811003_r1 4.31 7.42 0.24 103075581 135166383 80136859 108450426 131.13 135.33 0 0 0 0 0 0 62.45 81.56 4076489 1575168 4076489 1575168 70.96 77.81 4076489 1789702 4076489 1502806 11687564 11.34 5.40 0 17.39 0 1.12 0 0.37 0 0.00 0 24.30 0 2522253 0 43 0 41.49 0 1.16 0 0.01 0 1.14 0 0.00 0 284.54 0 0.36 0 183375 0 3398669 0 590872 0 38228 0 12447 0 0 0 825741 0 48 0 0 0 537 0 71688 0 1338 0 73611 0 56.83 0 1931381 0 9630 85930 8.923156801661 3398669.0 2522253.0 183375.0 590872.0 38228.0 12447.0 0.0 825741.0 1931381.0 74.2 5.4 17.4 1.1 0.4 0.0 24.3 56.8 43 43 43.00 38 146142767 26.7 22.1 22.1 29.1 0.0 36.2 24.6 smartseq 1070649 SRR2088563 SRP060416 SRS979879 SRX1082532 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811004: T86_P2_G8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811004 GSM1811004 T86_P2_G8_ILC2 86639926 2014882 2016-01-28 01:00:06 93322575 86639926 2014882 1 2014882 index:0,count:2014882,average:43,stdev:0 GSM1811004_r1 6.78 7.53 0.23 58603167 77676818 44255785 60825431 132.55 137.44 0 0 0 0 0 0 61.91 83.38 2418674 889666 2418674 889666 71.15 79.06 2418674 1022480 2418674 843486 6941025 11.84 6.09 0 18.37 0 1.15 0 0.26 0 0.00 0 27.26 0 1437135 0 43 0 41.48 0 1.14 0 0.01 0 1.10 0 0.00 0 241.79 0 0.37 0 122763 0 2014882 0 370175 0 23161 0 5237 0 0 0 549349 0 15 0 0 0 251 0 39846 0 790 0 40902 0 52.95 0 1066960 0 9059 47688 5.264157191743 2014882.0 1437135.0 122763.0 370175.0 23161.0 5237.0 0.0 549349.0 1066960.0 71.3 6.1 18.4 1.1 0.3 0.0 27.3 53.0 43 43 43.00 38 86639926 26.4 22.0 21.9 29.7 0.0 36.3 24.6 smartseq 1070667 SRR2088564 SRP060416 SRS980039 SRX1082533 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811005: T86_P2_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811005 GSM1811005 T86_P2_G9_NK 33550664 780248 2016-01-28 01:00:06 36348071 33550664 780248 1 780248 index:0,count:780248,average:43,stdev:0 GSM1811005_r1 5.87 6.07 0.19 25278021 33779467 20209686 27791055 133.63 137.51 0 0 0 0 0 0 63.49 80.36 957476 389929 957476 389929 70.65 76.29 957476 433890 957476 370187 2785051 11.02 4.33 0 16.53 0 1.22 0 0.44 0 0.00 0 19.62 0 614167 0 43 0 41.65 0 1.23 0 0.01 0 1.14 0 0.01 0 175.56 0 0.35 0 33816 0 780248 0 128952 0 9544 0 3427 0 0 0 153110 0 11 0 0 0 145 0 18010 0 341 0 18507 0 62.19 0 485215 0 7627 20909 2.741444866920 780248.0 614167.0 33816.0 128952.0 9544.0 3427.0 0.0 153110.0 485215.0 78.7 4.3 16.5 1.2 0.4 0.0 19.6 62.2 43 43 43.00 38 33550664 26.4 22.5 22.4 28.7 0.0 36.4 25.0 smartseq 1070684 SRR2088565 SRP060416 SRS980038 SRX1082534 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811006: T86_P2_H10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811006 GSM1811006 T86_P2_H10_ILC2 28586357 664799 2016-01-28 01:00:06 32530713 28586357 664799 1 664799 index:0,count:664799,average:43,stdev:0 GSM1811006_r1 4.65 8.6 0.25 18552495 23451958 14285361 18789872 126.41 131.53 0 0 0 0 0 0 57.68 76.47 765611 263747 765611 263747 64.72 73.24 765611 295950 765611 252624 2694215 14.52 6.78 0 16.90 0 1.09 0 0.34 0 0.00 0 29.79 0 457250 0 43 0 41.42 0 1.22 0 0.01 0 1.16 0 0.01 0 149.58 0 0.44 0 45103 0 664799 0 112341 0 7260 0 2241 0 0 0 198048 0 0 0 0 0 79 0 10335 0 239 0 10653 0 51.88 0 344909 0 4628 11963 2.584917891098 664799.0 457250.0 45103.0 112341.0 7260.0 2241.0 0.0 198048.0 344909.0 68.8 6.8 16.9 1.1 0.3 0.0 29.8 51.9 43 43 43.00 38 28586357 26.8 21.2 22.1 29.9 0.0 35.0 23.1 smartseq 1070699 SRR2088566 SRP060416 SRS980037 SRX1082535 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811007: T86_P2_H11_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811007 GSM1811007 T86_P2_H11_ILC2 185064389 4303823 2016-01-28 01:00:06 198574051 185064389 4303823 1 4303823 index:0,count:4303823,average:43,stdev:0 GSM1811007_r1 4.03 8.01 0.2 124951153 160269616 100305547 133211058 128.27 132.81 0 0 0 0 0 0 61.75 78.29 4821751 1887426 4821751 1887426 67.58 75.05 4821751 2065572 4821751 1809415 16786068 13.43 6.12 0 15.00 0 1.04 0 0.38 0 0.00 0 27.56 0 3056517 0 43 0 41.60 0 1.22 0 0.01 0 1.15 0 0.01 0 336.82 0 0.36 0 263394 0 4303823 0 645610 0 44767 0 16305 0 0 0 1186234 0 34 0 0 0 624 0 83507 0 1647 0 85812 0 56.02 0 2410907 0 9983 96175 9.633877591906 4303823.0 3056517.0 263394.0 645610.0 44767.0 16305.0 0.0 1186234.0 2410907.0 71.0 6.1 15.0 1.0 0.4 0.0 27.6 56.0 43 43 43.00 38 185064389 26.5 22.0 22.2 29.3 0.0 36.1 24.5 smartseq 1070715 SRR2088567 SRP060416 SRS980036 SRX1082536 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811008: T86_P2_H12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811008 GSM1811008 T86_P2_H12_ILC2 225554479 5245453 2016-01-28 01:00:06 240234477 225554479 5245453 1 5245453 index:0,count:5245453,average:43,stdev:0 GSM1811008_r1 6.62 6.56 0.19 166192684 222062831 131400003 181543917 133.62 138.16 0 0 0 0 0 0 62.49 80.07 6504287 2523289 6504287 2523289 70.32 76.44 6504287 2839425 6504287 2409018 19723610 11.87 4.82 0 16.90 0 0.96 0 0.37 0 0.00 0 21.69 0 4037689 0 43 0 41.70 0 1.12 0 0.01 0 1.12 0 0.01 0 356.29 0 0.35 0 252601 0 5245453 0 886308 0 50564 0 19502 0 0 0 1137698 0 12 0 0 0 912 0 117311 0 1998 0 120233 0 60.08 0 3151381 0 11782 139600 11.848582583602 5245453.0 4037689.0 252601.0 886308.0 50564.0 19502.0 0.0 1137698.0 3151381.0 77.0 4.8 16.9 1.0 0.4 0.0 21.7 60.1 43 43 43.00 38 225554479 26.5 22.3 22.4 28.8 0.0 36.3 24.9 smartseq 1070732 SRR2088568 SRP060416 SRS980035 SRX1082537 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811009: T86_P2_H2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811009 GSM1811009 T86_P2_H2_ILC2 54758952 1273464 2016-01-28 01:00:06 62143852 54758952 1273464 1 1273464 index:0,count:1273464,average:43,stdev:0 GSM1811009_r1 3.3 9.47 0.27 33385858 40992467 24697811 31640850 122.78 128.11 0 0 0 0 0 0 55.63 77.01 1461210 462098 1461210 462098 63.62 73.54 1461210 528454 1461210 441279 5236885 15.69 7.57 0 18.11 0 1.16 0 0.29 0 0.00 0 33.32 0 830702 0 43 0 41.16 0 1.24 0 0.00 0 1.13 0 0.01 0 416.77 0 0.46 0 96463 0 1273464 0 230644 0 14789 0 3671 0 0 0 424302 0 5 0 0 0 174 0 20183 0 509 0 20871 0 47.12 0 600058 0 6392 24102 3.770650813517 1273464.0 830702.0 96463.0 230644.0 14789.0 3671.0 0.0 424302.0 600058.0 65.2 7.6 18.1 1.2 0.3 0.0 33.3 47.1 43 43 43.00 38 54758952 26.6 21.1 22.0 30.3 0.0 34.9 22.8 smartseq 1070748 SRR2088569 SRP060416 SRS980033 SRX1082538 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811010: T86_P2_H5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811010 GSM1811010 T86_P2_H5_ILC2 106040365 2466055 2016-01-28 01:00:06 114567669 106040365 2466055 1 2466055 index:0,count:2466055,average:43,stdev:0 GSM1811010_r1 4.57 7.17 0.25 77445589 103114666 61242955 83884823 133.14 136.97 0 0 0 0 0 0 65.16 83.5 2980652 1233665 2980652 1233665 72.45 78.85 2980652 1371767 2980652 1164958 8160040 10.54 4.80 0 16.87 0 1.17 0 0.48 0 0.00 0 21.58 0 1893303 0 43 0 41.45 0 1.21 0 0.01 0 1.11 0 0.00 0 246.61 0 0.37 0 118406 0 2466055 0 415907 0 28810 0 11779 0 0 0 532163 0 22 0 0 0 448 0 56365 0 1008 0 57843 0 59.91 0 1477396 0 8690 65679 7.557997698504 2466055.0 1893303.0 118406.0 415907.0 28810.0 11779.0 0.0 532163.0 1477396.0 76.8 4.8 16.9 1.2 0.5 0.0 21.6 59.9 43 43 43.00 38 106040365 26.6 22.2 22.2 29.0 0.0 36.2 24.7 smartseq 1070860 SRR2088570 SRP060416 SRS980034 SRX1082539 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811011: T86_P2_H8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811011 GSM1811011 T86_P2_H8_ILC2 98977185 2301795 2016-01-28 01:00:06 107606862 98977185 2301795 1 2301795 index:0,count:2301795,average:43,stdev:0 GSM1811011_r1 6.19 6.82 0.18 71084809 93701629 56999023 77292101 131.82 135.6 0 0 0 0 0 0 63.65 80.49 2700728 1101190 2700728 1101190 70.74 76.51 2700728 1223825 2700728 1046789 8132189 11.44 5.26 0 15.72 0 1.01 0 0.34 0 0.00 0 23.49 0 1729952 0 43 0 41.66 0 1.14 0 0.01 0 1.14 0 0.00 0 360.28 0 0.36 0 121176 0 2301795 0 361861 0 23275 0 7928 0 0 0 540640 0 38 0 0 0 395 0 49667 0 875 0 50975 0 59.44 0 1368091 0 9570 57854 6.045350052247 2301795.0 1729952.0 121176.0 361861.0 23275.0 7928.0 0.0 540640.0 1368091.0 75.2 5.3 15.7 1.0 0.3 0.0 23.5 59.4 43 43 43.00 38 98977185 26.7 22.1 22.2 28.9 0.0 36.1 24.6 smartseq 1070875 SRR2088571 SRP060416 SRS980032 SRX1082540 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811012: T86_P2_H9_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811012 GSM1811012 T86_P2_H9_ILC2 32128224 747168 2016-01-28 01:00:06 34989491 32128224 747168 1 747168 index:0,count:747168,average:43,stdev:0 GSM1811012_r1 6.06 7.34 0.23 22103532 29122215 17404081 23728641 131.75 136.34 0 0 0 0 0 0 61.74 79.7 867149 333869 867149 333869 69.0 76.02 867149 373105 867149 318455 2829309 12.80 5.82 0 16.31 0 1.07 0 0.33 0 0.00 0 26.23 0 540752 0 43 0 41.55 0 1.19 0 0.01 0 1.16 0 0.00 0 149.43 0 0.36 0 43454 0 747168 0 121840 0 7985 0 2431 0 0 0 196000 0 5 0 0 0 132 0 14109 0 287 0 14533 0 56.07 0 418912 0 5732 16312 2.845778087927 747168.0 540752.0 43454.0 121840.0 7985.0 2431.0 0.0 196000.0 418912.0 72.4 5.8 16.3 1.1 0.3 0.0 26.2 56.1 43 43 43.00 38 32128224 26.9 21.8 21.8 29.4 0.0 36.2 24.6 smartseq 1070892 SRR2088572 SRP060416 SRS980029 SRX1082541 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811013: T86_P3_A11_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811013 GSM1811013 T86_P3_A11_ILC1 92289266 2146262 2016-01-28 01:00:06 102447623 92289266 2146262 1 2146262 index:0,count:2146262,average:43,stdev:0 GSM1811013_r1 7.01 7.7 0.19 57267461 73726286 45114574 60453925 128.74 134.0 0 0 0 0 0 0 57.71 74.64 2293545 808976 2293545 808976 64.41 71.87 2293545 902888 2293545 778882 9018001 15.75 7.47 0 14.81 0 0.95 0 0.35 0 0.00 0 33.39 0 1401747 0 43 0 41.63 0 1.11 0 0.01 0 1.14 0 0.01 0 227.25 0 0.37 0 160294 0 2146262 0 317939 0 20480 0 7450 0 0 0 716585 0 15 0 0 0 237 0 31745 0 716 0 32713 0 50.50 0 1083808 0 6919 36211 5.233559762972 2146262.0 1401747.0 160294.0 317939.0 20480.0 7450.0 0.0 716585.0 1083808.0 65.3 7.5 14.8 1.0 0.3 0.0 33.4 50.5 43 43 43.00 38 92289266 26.6 21.8 22.1 29.5 0.0 35.5 23.7 smartseq 1070908 SRR2088573 SRP060416 SRS980031 SRX1082542 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811014: T86_P3_A12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811014 GSM1811014 T86_P3_A12_ILC1 44716603 1039921 2016-01-28 01:00:06 51407684 44716603 1039921 1 1039921 index:0,count:1039921,average:43,stdev:0 GSM1811014_r1 6.43 7.78 0.19 28648427 36253457 21796095 28660385 126.55 131.49 0 0 0 0 0 0 55.57 74.42 1199770 390861 1199770 390861 63.71 71.05 1199770 448099 1199770 373174 4212283 14.70 7.03 0 17.13 0 1.04 0 0.32 0 0.00 0 31.01 0 703368 0 43 0 41.50 0 1.14 0 0.01 0 1.17 0 0.01 0 207.98 0 0.46 0 73123 0 1039921 0 178125 0 10811 0 3279 0 0 0 322463 0 9 0 0 0 112 0 16018 0 302 0 16441 0 50.51 0 525243 0 5987 18834 3.145815934525 1039921.0 703368.0 73123.0 178125.0 10811.0 3279.0 0.0 322463.0 525243.0 67.6 7.0 17.1 1.0 0.3 0.0 31.0 50.5 43 43 43.00 38 44716603 26.4 21.3 22.4 29.9 0.0 34.5 22.5 smartseq 1070923 SRR2088574 SRP060416 SRS980028 SRX1082543 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811015: T86_P3_A1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811015 GSM1811015 T86_P3_A1_ILC1 42006829 976903 2016-01-28 01:00:06 47819549 42006829 976903 1 976903 index:0,count:976903,average:43,stdev:0 GSM1811015_r1 6.48 7.7 0.25 25982094 32518390 19933632 25995290 125.16 130.41 0 0 0 0 0 0 56.04 74.42 1081155 356877 1081155 356877 63.29 71.1 1081155 403088 1081155 340917 4130144 15.90 7.49 0 16.11 0 1.10 0 0.37 0 0.00 0 33.34 0 636867 0 43 0 41.57 0 1.20 0 0.01 0 1.14 0 0.01 0 175.84 0 0.42 0 73140 0 976903 0 157351 0 10704 0 3585 0 0 0 325747 0 2 0 0 0 130 0 14335 0 343 0 14810 0 49.09 0 479516 0 5552 16942 3.051512968300 976903.0 636867.0 73140.0 157351.0 10704.0 3585.0 0.0 325747.0 479516.0 65.2 7.5 16.1 1.1 0.4 0.0 33.3 49.1 43 43 43.00 38 42006829 26.5 21.3 22.2 29.9 0.0 34.9 23.0 smartseq 1070939 SRR2088575 SRP060416 SRS980030 SRX1082544 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811016: T86_P3_A3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811016 GSM1811016 T86_P3_A3_ILC1 126422752 2940064 2016-01-28 01:00:06 137718748 126422752 2940064 1 2940064 index:0,count:2940064,average:43,stdev:0 GSM1811016_r1 7.9 6.47 0.19 82861980 109483621 63165883 86853285 132.13 137.5 0 0 0 0 0 0 59.49 79.12 3462016 1195511 3462016 1195511 69.46 75.93 3462016 1395885 3462016 1147317 10306355 12.44 6.77 0 16.96 0 1.05 0 0.36 0 0.00 0 30.23 0 2009760 0 43 0 41.80 0 1.15 0 0.01 0 1.15 0 0.00 0 235.21 0 0.35 0 199064 0 2940064 0 498743 0 30784 0 10663 0 0 0 888857 0 8 0 0 0 316 0 50459 0 991 0 51774 0 51.39 0 1511017 0 9417 61870 6.570032919189 2940064.0 2009760.0 199064.0 498743.0 30784.0 10663.0 0.0 888857.0 1511017.0 68.4 6.8 17.0 1.0 0.4 0.0 30.2 51.4 43 43 43.00 38 126422752 26.0 22.5 22.7 28.8 0.0 36.0 24.4 smartseq 1070953 SRR2088576 SRP060416 SRS980027 SRX1082545 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811017: T86_P3_A5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811017 GSM1811017 T86_P3_A5_ILC1 118249484 2749988 2016-01-28 01:00:06 130663126 118249484 2749988 1 2749988 index:0,count:2749988,average:43,stdev:0 GSM1811017_r1 4.76 5.48 0.18 84859690 110962301 65182758 87851624 130.76 134.78 0 0 0 0 0 0 59.58 78.26 3473301 1215642 3473301 1215642 69.88 74.15 3473301 1425680 3473301 1151741 9368160 11.04 5.47 0 17.71 0 0.92 0 0.39 0 0.00 0 24.50 0 2040227 0 43 0 41.96 0 1.15 0 0.01 0 1.13 0 0.01 0 330.00 0 0.37 0 150386 0 2749988 0 486945 0 25231 0 10800 0 0 0 673730 0 15 0 0 0 451 0 62160 0 861 0 63487 0 56.48 0 1553282 0 12122 76140 6.281141725788 2749988.0 2040227.0 150386.0 486945.0 25231.0 10800.0 0.0 673730.0 1553282.0 74.2 5.5 17.7 0.9 0.4 0.0 24.5 56.5 43 43 43.00 38 118249484 26.0 23.0 23.4 27.7 0.0 35.7 24.3 smartseq 1070970 SRR2088577 SRP060416 SRS980026 SRX1082546 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811018: T86_P3_A8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811018 GSM1811018 T86_P3_A8_ILC1 68668291 1596937 2016-01-28 01:00:06 76339075 68668291 1596937 1 1596937 index:0,count:1596937,average:43,stdev:0 GSM1811018_r1 6.09 6.6 0.17 46359768 60649008 35922472 48548151 130.82 135.15 0 0 0 0 0 0 58.82 76.87 1856732 660124 1856732 660124 67.7 72.95 1856732 759786 1856732 626401 6053122 13.06 6.36 0 16.50 0 0.97 0 0.38 0 0.00 0 28.38 0 1122294 0 43 0 41.83 0 1.18 0 0.01 0 1.10 0 0.01 0 205.32 0 0.37 0 101502 0 1596937 0 263570 0 15483 0 6002 0 0 0 453158 0 11 0 0 0 211 0 29078 0 506 0 29806 0 53.77 0 858724 0 7925 35561 4.487192429022 1596937.0 1122294.0 101502.0 263570.0 15483.0 6002.0 0.0 453158.0 858724.0 70.3 6.4 16.5 1.0 0.4 0.0 28.4 53.8 43 43 43.00 38 68668291 26.3 22.4 22.8 28.5 0.0 35.7 24.1 smartseq 1070986 SRR2088578 SRP060416 SRS980025 SRX1082547 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811019: T86_P3_A9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811019 GSM1811019 T86_P3_A9_ILC1 25695897 597579 2016-01-28 01:00:06 28073435 25695897 597579 1 597579 index:0,count:597579,average:43,stdev:0 GSM1811019_r1 7.79 6.2 0.21 17493732 23411528 13317609 18464418 133.83 138.65 0 0 0 0 0 0 60.86 80.91 719135 257728 719135 257728 71.01 76.8 719135 300709 719135 244649 1857066 10.62 6.19 0 17.56 0 1.04 0 0.33 0 0.00 0 27.76 0 423500 0 43 0 41.81 0 1.19 0 0.01 0 1.14 0 0.00 0 107.56 0 0.34 0 36989 0 597579 0 104955 0 6202 0 1994 0 0 0 165883 0 4 0 0 0 93 0 11486 0 201 0 11784 0 53.31 0 318545 0 5634 14009 2.486510472133 597579.0 423500.0 36989.0 104955.0 6202.0 1994.0 0.0 165883.0 318545.0 70.9 6.2 17.6 1.0 0.3 0.0 27.8 53.3 43 43 43.00 38 25695897 26.1 22.7 22.7 28.5 0.0 36.2 24.8 smartseq 1071002 SRR2088579 SRP060416 SRS980024 SRX1082548 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811020: T86_P3_B12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811020 GSM1811020 T86_P3_B12_ILC1 382470208 8894656 2016-01-28 01:00:06 402910247 382470208 8894656 1 8894656 index:0,count:8894656,average:43,stdev:0 GSM1811020_r1 8.26 6.35 0.22 247997430 330468598 191104206 262855283 133.25 137.55 0 0 0 0 0 0 61.3 80.6 9905364 3690200 9905364 3690200 70.96 76.91 9905364 4271893 9905364 3521215 28187291 11.37 6.97 0 16.21 0 0.92 0 0.33 0 0.00 0 31.06 0 6020116 0 43 0 41.74 0 1.12 0 0.01 0 1.12 0 0.00 0 264.63 0 0.34 0 620135 0 8894656 0 1441767 0 82019 0 29454 0 0 0 2763067 0 17 0 0 0 1247 0 163812 0 3143 0 168219 0 51.47 0 4578349 0 12398 202595 16.340942087433 8894656.0 6020116.0 620135.0 1441767.0 82019.0 29454.0 0.0 2763067.0 4578349.0 67.7 7.0 16.2 0.9 0.3 0.0 31.1 51.5 43 43 43.00 38 382470208 25.8 22.8 22.8 28.6 0.0 36.1 24.6 smartseq 1071114 SRR2088580 SRP060416 SRS980023 SRX1082549 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811021: T86_P3_B1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811021 GSM1811021 T86_P3_B1_ILC1 187606377 4362939 2016-01-28 01:00:06 200886300 187606377 4362939 1 4362939 index:0,count:4362939,average:43,stdev:0 GSM1811021_r1 7.21 6.74 0.2 118257916 152907243 90939224 122242971 129.3 134.42 0 0 0 0 0 0 55.48 73.3 4912257 1597460 4912257 1597460 63.87 69.92 4912257 1839048 4912257 1523889 18199395 15.39 7.30 0 16.04 0 1.01 0 0.37 0 0.00 0 32.62 0 2879275 0 43 0 41.73 0 1.18 0 0.01 0 1.11 0 0.01 0 253.33 0 0.33 0 318339 0 4362939 0 699846 0 44223 0 16225 0 0 0 1423216 0 36 0 0 0 559 0 66685 0 1617 0 68897 0 49.95 0 2179429 0 8540 79904 9.356440281030 4362939.0 2879275.0 318339.0 699846.0 44223.0 16225.0 0.0 1423216.0 2179429.0 66.0 7.3 16.0 1.0 0.4 0.0 32.6 50.0 43 43 43.00 38 187606377 26.1 22.4 22.4 29.1 0.0 36.2 24.5 smartseq 1071130 SRR2088581 SRP060416 SRS980022 SRX1082550 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811022: T86_P3_B3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811022 GSM1811022 T86_P3_B3_ILC1 187971748 4371436 2016-01-28 01:00:06 201554807 187971748 4371436 1 4371436 index:0,count:4371436,average:43,stdev:0 GSM1811022_r1 7.33 6.07 0.18 125362024 164617171 98500232 134130201 131.31 136.17 0 0 0 0 0 0 58.5 75.41 5029614 1775245 5029614 1775245 66.72 72.21 5029614 2024569 5029614 1700052 17370159 13.86 6.50 0 15.56 0 1.02 0 0.41 0 0.00 0 29.15 0 3034490 0 43 0 41.84 0 1.16 0 0.01 0 1.11 0 0.00 0 314.74 0 0.33 0 284055 0 4371436 0 680303 0 44689 0 17913 0 0 0 1274344 0 48 0 0 0 615 0 71213 0 1393 0 73269 0 53.85 0 2354187 0 9769 86395 8.843791585628 4371436.0 3034490.0 284055.0 680303.0 44689.0 17913.0 0.0 1274344.0 2354187.0 69.4 6.5 15.6 1.0 0.4 0.0 29.2 53.9 43 43 43.00 38 187971748 26.0 22.6 22.7 28.6 0.0 36.2 24.8 smartseq 1071146 SRR2088582 SRP060416 SRS980021 SRX1082551 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811023: T86_P3_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811023 GSM1811023 T86_P3_B4_NK 161962682 3766574 2016-01-28 01:00:06 173709587 161962682 3766574 1 3766574 index:0,count:3766574,average:43,stdev:0 GSM1811023_r1 8.58 6.26 0.2 103466908 136014203 83585697 112944273 131.46 135.12 0 0 0 0 0 0 59.16 74.16 3869857 1481723 3869857 1481723 65.48 70.47 3869857 1639947 3869857 1408068 15275249 14.76 7.20 0 13.45 0 0.85 0 0.45 0 0.00 0 32.19 0 2504691 0 43 0 41.83 0 1.13 0 0.01 0 1.12 0 0.00 0 330.72 0 0.32 0 271175 0 3766574 0 506556 0 32177 0 17102 0 0 0 1212604 0 46 0 0 0 506 0 66869 0 1248 0 68669 0 53.05 0 1998135 0 10259 77532 7.557461740910 3766574.0 2504691.0 271175.0 506556.0 32177.0 17102.0 0.0 1212604.0 1998135.0 66.5 7.2 13.4 0.9 0.5 0.0 32.2 53.0 43 43 43.00 38 161962682 25.9 22.8 22.8 28.5 0.0 36.3 24.9 smartseq 1071162 SRR2088583 SRP060416 SRS980020 SRX1082552 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811024: T86_P3_B6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811024 GSM1811024 T86_P3_B6_ILC1 173200603 4027921 2016-01-28 01:00:06 189724507 173200603 4027921 1 4027921 index:0,count:4027921,average:43,stdev:0 GSM1811024_r1 7.79 6.02 0.2 112001595 147397316 85393695 116440379 131.6 136.36 0 0 0 0 0 0 59.68 79.25 4637731 1619623 4637731 1619623 69.8 75.61 4637731 1894360 4637731 1545194 13172300 11.76 7.05 0 16.64 0 0.86 0 0.34 0 0.00 0 31.43 0 2713792 0 43 0 41.79 0 1.12 0 0.01 0 1.14 0 0.01 0 268.53 0 0.37 0 283940 0 4027921 0 670199 0 34446 0 13751 0 0 0 1265932 0 18 0 0 0 587 0 71013 0 1268 0 72886 0 50.74 0 2043593 0 11330 88849 7.841924095322 4027921.0 2713792.0 283940.0 670199.0 34446.0 13751.0 0.0 1265932.0 2043593.0 67.4 7.0 16.6 0.9 0.3 0.0 31.4 50.7 43 43 43.00 38 173200603 26.2 22.5 22.9 28.5 0.0 35.7 24.2 smartseq 1071178 SRR2088584 SRP060416 SRS980019 SRX1082553 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811025: T86_P3_B7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811025 GSM1811025 T86_P3_B7_ILC1 220089136 5118352 2016-01-28 01:00:06 234529088 220089136 5118352 1 5118352 index:0,count:5118352,average:43,stdev:0 GSM1811025_r1 8.32 5.75 0.19 157735264 215440626 121502132 171134750 136.58 140.85 0 0 0 0 0 0 59.27 77.73 6278752 2256096 6278752 2256096 70.15 74.09 6278752 2670365 6278752 2150396 18239663 11.56 5.41 0 17.67 0 1.06 0 0.42 0 0.00 0 24.15 0 3806672 0 43 0 41.86 0 1.15 0 0.01 0 1.13 0 0.00 0 83.00 0 0.33 0 276984 0 5118352 0 904329 0 54004 0 21554 0 0 0 1236122 0 43 0 0 0 845 0 93825 0 1724 0 96437 0 56.70 0 2902343 0 13107 119622 9.126573586633 5118352.0 3806672.0 276984.0 904329.0 54004.0 21554.0 0.0 1236122.0 2902343.0 74.4 5.4 17.7 1.1 0.4 0.0 24.2 56.7 43 43 43.00 38 220089136 25.9 22.8 22.8 28.4 0.0 36.3 24.9 smartseq 1071194 SRR2088585 SRP060416 SRS979883 SRX1082554 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811026: T86_P3_B8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811026 GSM1811026 T86_P3_B8_ILC1 59000902 1372114 2016-01-28 01:00:06 67097692 59000902 1372114 1 1372114 index:0,count:1372114,average:43,stdev:0 GSM1811026_r1 7.3 7.56 0.2 36134173 46553319 27787646 37255727 128.83 134.07 0 0 0 0 0 0 56.61 74.92 1481498 500844 1481498 500844 64.52 71.59 1481498 570865 1481498 478553 5083247 14.07 7.57 0 15.77 0 0.98 0 0.35 0 0.00 0 34.19 0 884800 0 43 0 41.57 0 1.17 0 0.01 0 1.15 0 0.00 0 235.22 0 0.42 0 103917 0 1372114 0 216321 0 13416 0 4778 0 0 0 469120 0 23 0 0 0 150 0 20968 0 452 0 21593 0 48.72 0 668479 0 7321 24781 3.384920092883 1372114.0 884800.0 103917.0 216321.0 13416.0 4778.0 0.0 469120.0 668479.0 64.5 7.6 15.8 1.0 0.3 0.0 34.2 48.7 43 43 43.00 38 59000902 26.4 21.8 22.6 29.2 0.0 34.9 23.0 smartseq 1071211 SRR2088586 SRP060416 SRS980018 SRX1082555 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811027: T86_P3_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811027 GSM1811027 T86_P3_B9_NK 49259037 1145559 2016-01-28 01:00:06 53519546 49259037 1145559 1 1145559 index:0,count:1145559,average:43,stdev:0 GSM1811027_r1 7.69 5.64 0.2 34526157 45651724 27454857 37381776 132.22 136.16 0 0 0 0 0 0 58.23 74.04 1310437 486050 1310437 486050 66.22 70.42 1310437 552749 1310437 462316 4763760 13.80 5.74 0 15.56 0 1.09 0 0.50 0 0.00 0 25.54 0 834721 0 43 0 41.82 0 1.19 0 0.01 0 1.12 0 0.00 0 187.46 0 0.33 0 65741 0 1145559 0 178231 0 12533 0 5744 0 0 0 292561 0 10 0 0 0 172 0 24458 0 350 0 24990 0 57.31 0 656490 0 9676 28730 2.969202149649 1145559.0 834721.0 65741.0 178231.0 12533.0 5744.0 0.0 292561.0 656490.0 72.9 5.7 15.6 1.1 0.5 0.0 25.5 57.3 43 43 43.00 38 49259037 26.2 22.7 22.7 28.4 0.0 36.3 24.9 smartseq 1071227 SRR2088587 SRP060416 SRS980017 SRX1082556 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811028: T86_P3_C10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811028 GSM1811028 T86_P3_C10_ILC1 44941966 1045162 2016-01-28 01:00:06 50071681 44941966 1045162 1 1045162 index:0,count:1045162,average:43,stdev:0 GSM1811028_r1 5.93 7.75 0.22 26455927 33373179 20404933 26722544 126.15 130.96 0 0 0 0 0 0 55.97 73.87 1073727 362543 1073727 362543 63.62 70.52 1073727 412150 1073727 346117 4097959 15.49 8.23 0 15.02 0 0.95 0 0.31 0 0.00 0 36.76 0 647799 0 43 0 41.58 0 1.16 0 0.01 0 1.12 0 0.01 0 198.03 0 0.36 0 86037 0 1045162 0 157018 0 9940 0 3188 0 0 0 384235 0 5 0 0 0 97 0 15956 0 346 0 16404 0 46.96 0 490781 0 5729 18578 3.242799790539 1045162.0 647799.0 86037.0 157018.0 9940.0 3188.0 0.0 384235.0 490781.0 62.0 8.2 15.0 1.0 0.3 0.0 36.8 47.0 43 43 43.00 38 44941966 26.2 22.1 22.4 29.2 0.0 35.6 23.8 smartseq 1071242 SRR2088588 SRP060416 SRS980016 SRX1082557 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811029: T86_P3_C11_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811029 GSM1811029 T86_P3_C11_ILC1 159619053 3712071 2016-01-28 01:00:06 171345705 159619053 3712071 1 3712071 index:0,count:3712071,average:43,stdev:0 GSM1811029_r1 8.4 5.8 0.19 109598828 147177368 85076926 117864731 134.29 138.54 0 0 0 0 0 0 62.1 80.87 4325800 1645477 4325800 1645477 71.52 76.84 4325800 1894929 4325800 1563503 11926496 10.88 6.16 0 16.56 0 0.92 0 0.31 0 0.00 0 27.40 0 2649541 0 43 0 41.81 0 1.12 0 0.01 0 1.12 0 0.01 0 272.72 0 0.33 0 228630 0 3712071 0 614869 0 34241 0 11360 0 0 0 1016929 0 40 0 0 0 564 0 73467 0 1348 0 75419 0 54.81 0 2034672 0 11952 90255 7.551455823293 3712071.0 2649541.0 228630.0 614869.0 34241.0 11360.0 0.0 1016929.0 2034672.0 71.4 6.2 16.6 0.9 0.3 0.0 27.4 54.8 43 43 43.00 38 159619053 25.8 22.9 22.9 28.3 0.0 36.3 25.0 smartseq 1071258 SRR2088589 SRP060416 SRS980015 SRX1082558 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811030: T86_P3_C12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811030 GSM1811030 T86_P3_C12_ILC1 146444197 3405679 2016-01-28 01:00:06 158711523 146444197 3405679 1 3405679 index:0,count:3405679,average:43,stdev:0 GSM1811030_r1 7.46 6.64 0.23 96290311 128000857 75525713 103586483 132.93 137.15 0 0 0 0 0 0 60.69 78.36 3761147 1416801 3761147 1416801 69.38 74.77 3761147 1619677 3761147 1351913 11712821 12.16 6.80 0 15.46 0 0.91 0 0.34 0 0.00 0 30.20 0 2334499 0 43 0 41.77 0 1.14 0 0.01 0 1.10 0 0.00 0 235.78 0 0.33 0 231608 0 3405679 0 526443 0 31104 0 11546 0 0 0 1028530 0 29 0 0 0 390 0 61107 0 1151 0 62677 0 53.09 0 1808056 0 9830 73645 7.491861648016 3405679.0 2334499.0 231608.0 526443.0 31104.0 11546.0 0.0 1028530.0 1808056.0 68.5 6.8 15.5 0.9 0.3 0.0 30.2 53.1 43 43 43.00 38 146444197 26.0 22.7 22.7 28.6 0.0 36.1 24.6 smartseq 1071370 SRR2088590 SRP060416 SRS980012 SRX1082559 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811031: T86_P3_C1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811031 GSM1811031 T86_P3_C1_ILC1 148516969 3453883 2016-01-28 01:00:06 159448427 148516969 3453883 1 3453883 index:0,count:3453883,average:43,stdev:0 GSM1811031_r1 5.76 6.77 0.19 93422037 120166658 71325899 95259283 128.63 133.55 0 0 0 0 0 0 55.87 74.26 3872472 1269285 3872472 1269285 65.02 70.47 3872472 1477030 3872472 1204522 13153728 14.08 7.33 0 16.29 0 1.03 0 0.37 0 0.00 0 32.83 0 2271735 0 43 0 41.73 0 1.14 0 0.01 0 1.11 0 0.00 0 259.04 0 0.32 0 253133 0 3453883 0 562515 0 35462 0 12786 0 0 0 1133900 0 12 0 0 0 449 0 54066 0 1171 0 55698 0 49.49 0 1709220 0 8278 67838 8.194974631554 3453883.0 2271735.0 253133.0 562515.0 35462.0 12786.0 0.0 1133900.0 1709220.0 65.8 7.3 16.3 1.0 0.4 0.0 32.8 49.5 43 43 43.00 38 148516969 25.9 22.5 22.5 29.0 0.0 36.2 24.6 smartseq 1071386 SRR2088591 SRP060416 SRS979882 SRX1082560 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811032: T86_P3_C2_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811032 GSM1811032 T86_P3_C2_ILC1 40335634 938038 2016-01-28 01:00:06 45405187 40335634 938038 1 938038 index:0,count:938038,average:43,stdev:0 GSM1811032_r1 5.19 6.81 0.2 24829499 31958637 19342565 25643839 128.71 132.58 0 0 0 0 0 0 59.65 77.59 984477 359724 984477 359724 67.68 73.47 984477 408161 984477 340656 3233292 13.02 7.79 0 14.86 0 0.89 0 0.30 0 0.00 0 34.52 0 603032 0 43 0 41.72 0 1.19 0 0.01 0 1.08 0 0.00 0 33.11 0 0.40 0 73066 0 938038 0 139389 0 8374 0 2833 0 0 0 323799 0 12 0 0 0 113 0 17709 0 268 0 18102 0 49.43 0 463643 0 6766 20526 3.033697901271 938038.0 603032.0 73066.0 139389.0 8374.0 2833.0 0.0 323799.0 463643.0 64.3 7.8 14.9 0.9 0.3 0.0 34.5 49.4 43 43 43.00 38 40335634 26.5 22.1 22.8 28.6 0.0 35.4 23.8 smartseq 1071402 SRR2088592 SRP060416 SRS979881 SRX1082561 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811033: T86_P3_C4_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811033 GSM1811033 T86_P3_C4_ILC1 129563902 3013114 2016-01-28 01:00:06 139639812 129563902 3013114 1 3013114 index:0,count:3013114,average:43,stdev:0 GSM1811033_r1 7.24 6.06 0.19 85677098 114629064 67228577 93137642 133.79 138.54 0 0 0 0 0 0 62.57 80.68 3399063 1296432 3399063 1296432 71.48 77.17 3399063 1481160 3399063 1239982 9263184 10.81 6.70 0 15.44 0 0.92 0 0.33 0 0.00 0 29.98 0 2072043 0 43 0 41.84 0 1.13 0 0.01 0 1.11 0 0.00 0 319.04 0 0.32 0 201829 0 3013114 0 465140 0 27668 0 10077 0 0 0 903326 0 22 0 0 0 405 0 56963 0 1002 0 58392 0 53.33 0 1606903 0 10678 68528 6.417681213710 3013114.0 2072043.0 201829.0 465140.0 27668.0 10077.0 0.0 903326.0 1606903.0 68.8 6.7 15.4 0.9 0.3 0.0 30.0 53.3 43 43 43.00 38 129563902 25.8 22.9 22.9 28.5 0.0 36.3 24.8 smartseq 1071420 SRR2088593 SRP060416 SRS980014 SRX1082562 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811034: T86_P3_C5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811034 GSM1811034 T86_P3_C5_ILC1 144433173 3358911 2016-01-28 01:00:06 156566267 144433173 3358911 1 3358911 index:0,count:3358911,average:43,stdev:0 GSM1811034_r1 6.55 6.33 0.19 91787460 119602324 74234755 99663538 130.3 134.25 0 0 0 0 0 0 60.87 76.13 3469194 1349682 3469194 1349682 68.2 73.3 3469194 1512083 3469194 1299512 11893110 12.96 7.33 0 13.23 0 0.89 0 0.38 0 0.00 0 32.72 0 2217152 0 43 0 41.87 0 1.18 0 0.01 0 1.17 0 0.01 0 262.87 0 0.33 0 246067 0 3358911 0 444298 0 29744 0 12931 0 0 0 1099084 0 27 0 0 0 459 0 57342 0 1091 0 58919 0 52.78 0 1772854 0 9680 67699 6.993698347107 3358911.0 2217152.0 246067.0 444298.0 29744.0 12931.0 0.0 1099084.0 1772854.0 66.0 7.3 13.2 0.9 0.4 0.0 32.7 52.8 43 43 43.00 38 144433173 26.0 22.9 23.0 28.1 0.0 36.1 24.7 smartseq 1071436 SRR2088594 SRP060416 SRS980013 SRX1082563 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811035: T86_P3_C6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811035 GSM1811035 T86_P3_C6_ILC1 152913977 3556139 2016-01-28 01:00:06 165585017 152913977 3556139 1 3556139 index:0,count:3556139,average:43,stdev:0 GSM1811035_r1 9.2 6.37 0.19 101487426 138057383 81484768 114327933 136.03 140.31 0 0 0 0 0 0 62.43 78.7 3790245 1532985 3790245 1532985 70.02 75.4 3790245 1719391 3790245 1468604 12336101 12.16 6.68 0 14.27 0 0.81 0 0.34 0 0.00 0 29.80 0 2455399 0 43 0 41.83 0 1.15 0 0.01 0 1.13 0 0.00 0 251.02 0 0.33 0 237509 0 3556139 0 507564 0 28688 0 12215 0 0 0 1059837 0 11 0 0 0 457 0 65399 0 1155 0 67022 0 54.77 0 1947835 0 10270 77107 7.507984420643 3556139.0 2455399.0 237509.0 507564.0 28688.0 12215.0 0.0 1059837.0 1947835.0 69.0 6.7 14.3 0.8 0.3 0.0 29.8 54.8 43 43 43.00 38 152913977 26.0 22.8 22.9 28.3 0.0 36.1 24.7 smartseq 1071451 SRR2088595 SRP060416 SRS980011 SRX1082564 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811036: T86_P3_C7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811036 GSM1811036 T86_P3_C7_ILC1 175316547 4077129 2016-01-28 01:00:06 187819036 175316547 4077129 1 4077129 index:0,count:4077129,average:43,stdev:0 GSM1811036_r1 8.64 6.04 0.16 118948250 161466419 93229034 130816270 135.75 140.32 0 0 0 0 0 0 63.23 81.58 4638906 1818250 4638906 1818250 72.27 77.61 4638906 2078182 4638906 1729816 12471511 10.48 6.33 0 15.86 0 0.97 0 0.32 0 0.00 0 28.18 0 2875460 0 43 0 41.83 0 1.15 0 0.01 0 1.13 0 0.00 0 299.54 0 0.32 0 258254 0 4077129 0 646690 0 39728 0 13073 0 0 0 1148868 0 30 0 0 0 625 0 87156 0 1340 0 89151 0 54.67 0 2228770 0 12231 105378 8.615648761344 4077129.0 2875460.0 258254.0 646690.0 39728.0 13073.0 0.0 1148868.0 2228770.0 70.5 6.3 15.9 1.0 0.3 0.0 28.2 54.7 43 43 43.00 38 175316547 25.8 23.0 23.0 28.3 0.0 36.3 24.9 smartseq 1071467 SRR2088596 SRP060416 SRS980010 SRX1082565 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811037: T86_P3_C8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811037 GSM1811037 T86_P3_C8_ILC1 80510018 1872326 2016-01-28 01:00:06 87469618 80510018 1872326 1 1872326 index:0,count:1872326,average:43,stdev:0 GSM1811037_r1 8.4 7.11 0.21 46049450 59437731 34391194 46430561 129.07 135.01 0 0 0 0 0 0 55.84 76.1 1987847 629996 1987847 629996 65.74 73.36 1987847 741682 1987847 607272 6586519 14.30 8.62 0 16.04 0 1.03 0 0.35 0 0.00 0 38.37 0 1128154 0 43 0 41.54 0 1.13 0 0.01 0 1.10 0 0.00 0 187.23 0 0.35 0 161483 0 1872326 0 300335 0 19225 0 6556 0 0 0 718391 0 12 0 0 0 172 0 23891 0 631 0 24706 0 44.21 0 827819 0 6624 29669 4.479015700483 1872326.0 1128154.0 161483.0 300335.0 19225.0 6556.0 0.0 718391.0 827819.0 60.3 8.6 16.0 1.0 0.4 0.0 38.4 44.2 43 43 43.00 38 80510018 25.9 22.4 22.4 29.2 0.0 36.1 24.3 smartseq 1071483 SRR2088597 SRP060416 SRS980009 SRX1082566 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811038: T86_P3_C9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811038 GSM1811038 T86_P3_C9_ILC1 35171678 817946 2016-01-28 01:00:06 38271660 35171678 817946 1 817946 index:0,count:817946,average:43,stdev:0 GSM1811038_r1 10.49 6.4 0.2 22924737 31148373 18376095 25908618 135.87 140.99 0 0 0 0 0 0 61.29 77.57 877338 341287 877338 341287 68.11 74.33 877338 379225 877338 327029 2882545 12.57 6.89 0 14.29 0 0.86 0 0.33 0 0.00 0 30.73 0 556823 0 43 0 41.77 0 1.14 0 0.01 0 1.13 0 0.01 0 196.31 0 0.32 0 56383 0 817946 0 116873 0 7056 0 2708 0 0 0 251359 0 8 0 0 0 111 0 13415 0 282 0 13816 0 53.79 0 439950 0 6211 15276 2.459507325712 817946.0 556823.0 56383.0 116873.0 7056.0 2708.0 0.0 251359.0 439950.0 68.1 6.9 14.3 0.9 0.3 0.0 30.7 53.8 43 43 43.00 38 35171678 26.2 22.4 22.4 29.0 0.0 36.3 24.7 smartseq 1071499 SRR2088598 SRP060416 SRS980008 SRX1082567 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811039: T86_P3_D10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811039 GSM1811039 T86_P3_D10_ILC1 24815644 577108 2016-01-28 01:00:06 27049009 24815644 577108 1 577108 index:0,count:577108,average:43,stdev:0 GSM1811039_r1 9.09 6.4 0.21 16473062 22456570 12930611 18144865 136.32 140.32 0 0 0 0 0 0 64.01 82.47 639170 255289 639170 255289 73.58 78.91 639170 293461 639170 244263 1682286 10.21 6.64 0 15.47 0 0.90 0 0.27 0 0.00 0 29.73 0 398834 0 43 0 41.77 0 1.13 0 0.01 0 1.13 0 0.00 0 115.42 0 0.32 0 38328 0 577108 0 89282 0 5178 0 1535 0 0 0 171561 0 8 0 0 0 80 0 11221 0 175 0 11484 0 53.64 0 309552 0 5546 13131 2.367652362063 577108.0 398834.0 38328.0 89282.0 5178.0 1535.0 0.0 171561.0 309552.0 69.1 6.6 15.5 0.9 0.3 0.0 29.7 53.6 43 43 43.00 38 24815644 25.8 23.0 23.0 28.2 0.0 36.3 25.0 smartseq 1071515 SRR2088599 SRP060416 SRS980007 SRX1082568 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811040: T86_P3_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811040 GSM1811040 T86_P3_D11_NK 54544984 1268488 2016-01-28 01:00:06 58999495 54544984 1268488 1 1268488 index:0,count:1268488,average:43,stdev:0 GSM1811040_r1 9.28 6.13 0.16 35601014 47844832 28269763 39238115 134.39 138.8 0 0 0 0 0 0 62.15 79.25 1367819 537231 1367819 537231 69.57 75.36 1367819 601417 1367819 510875 4436806 12.46 6.85 0 14.71 0 0.93 0 0.33 0 0.00 0 30.59 0 864431 0 43 0 41.70 0 1.12 0 0.01 0 1.12 0 0.00 0 326.18 0 0.32 0 86930 0 1268488 0 186550 0 11781 0 4200 0 0 0 388076 0 11 0 0 0 193 0 24690 0 441 0 25335 0 53.44 0 677881 0 7912 28836 3.644590495450 1268488.0 864431.0 86930.0 186550.0 11781.0 4200.0 0.0 388076.0 677881.0 68.1 6.9 14.7 0.9 0.3 0.0 30.6 53.4 43 43 43.00 38 54544984 25.9 22.9 22.8 28.4 0.0 36.4 25.1 smartseq 1073163 SRR2088600 SRP060416 SRS980005 SRX1082569 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811041: T86_P3_D12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811041 GSM1811041 T86_P3_D12_ILC1 60495238 1406866 2016-01-28 01:00:06 65511991 60495238 1406866 1 1406866 index:0,count:1406866,average:43,stdev:0 GSM1811041_r1 7.87 7.46 0.23 31296604 40095505 23919801 31801156 128.11 132.95 0 0 0 0 0 0 57.51 76.57 1293131 441012 1293131 441012 65.62 72.73 1293131 503212 1293131 418867 4657672 14.88 10.06 0 13.57 0 0.82 0 0.25 0 0.00 0 44.41 0 766908 0 43 0 41.53 0 1.13 0 0.01 0 1.15 0 0.01 0 337.65 0 0.34 0 141497 0 1406866 0 190962 0 11591 0 3567 0 0 0 624800 0 11 0 0 0 135 0 18410 0 468 0 19024 0 40.94 0 575946 0 5986 22059 3.685098563314 1406866.0 766908.0 141497.0 190962.0 11591.0 3567.0 0.0 624800.0 575946.0 54.5 10.1 13.6 0.8 0.3 0.0 44.4 40.9 43 43 43.00 38 60495238 25.9 22.7 22.7 28.8 0.0 36.3 24.8 smartseq 1073178 SRR2088601 SRP060416 SRS980006 SRX1082570 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811042: T86_P3_D1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811042 GSM1811042 T86_P3_D1_ILC1 61336662 1426434 2016-01-28 01:00:06 66332740 61336662 1426434 1 1426434 index:0,count:1426434,average:43,stdev:0 GSM1811042_r1 9.16 5.45 0.17 41772326 57002628 32551203 45864101 136.46 140.9 0 0 0 0 0 0 64.23 83.3 1641298 648065 1641298 648065 73.33 78.73 1641298 739829 1641298 612518 3694385 8.84 6.26 0 16.19 0 0.90 0 0.37 0 0.00 0 28.00 0 1008928 0 43 0 41.84 0 1.13 0 0.01 0 1.14 0 0.00 0 285.29 0 0.32 0 89240 0 1426434 0 230919 0 12879 0 5296 0 0 0 399331 0 13 0 0 0 228 0 31931 0 490 0 32662 0 54.54 0 778009 0 9914 38171 3.850211821666 1426434.0 1008928.0 89240.0 230919.0 12879.0 5296.0 0.0 399331.0 778009.0 70.7 6.3 16.2 0.9 0.4 0.0 28.0 54.5 43 43 43.00 38 61336662 25.7 23.1 23.1 28.1 0.0 36.4 25.2 smartseq 1073193 SRR2088602 SRP060416 SRS979884 SRX1082571 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811043: T86_P3_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811043 GSM1811043 T86_P3_D2_NK 24259525 564175 2016-01-28 01:00:06 26391125 24259525 564175 1 564175 index:0,count:564175,average:43,stdev:0 GSM1811043_r1 8.65 6.39 0.18 15580943 20246386 12534070 16795633 129.94 134.0 0 0 0 0 0 0 58.06 73.18 590378 219627 590378 219627 64.19 69.79 590378 242845 590378 209442 2443333 15.68 7.07 0 13.86 0 0.89 0 0.42 0 0.00 0 31.64 0 378303 0 43 0 41.76 0 1.13 0 0.01 0 1.11 0 0.01 0 106.90 0 0.32 0 39898 0 564175 0 78179 0 5021 0 2360 0 0 0 178491 0 13 0 0 0 66 0 9873 0 160 0 10112 0 53.20 0 300124 0 4716 11070 2.347328244275 564175.0 378303.0 39898.0 78179.0 5021.0 2360.0 0.0 178491.0 300124.0 67.1 7.1 13.9 0.9 0.4 0.0 31.6 53.2 43 43 43.00 38 24259525 26.1 22.6 22.6 28.8 0.0 36.4 24.9 smartseq 1073210 SRR2088603 SRP060416 SRS980004 SRX1082572 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811044: T86_P3_D3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811044 GSM1811044 T86_P3_D3_ILC1 59744243 1389401 2016-01-28 01:00:06 64499266 59744243 1389401 1 1389401 index:0,count:1389401,average:43,stdev:0 GSM1811044_r1 8.89 6.24 0.17 38526316 51714285 31250469 43252568 134.23 138.41 0 0 0 0 0 0 61.28 76.52 1435564 571695 1435564 571695 67.8 73.09 1435564 632477 1435564 546041 5122583 13.30 7.04 0 13.37 0 0.86 0 0.40 0 0.00 0 31.59 0 932923 0 43 0 41.83 0 1.11 0 0.01 0 1.12 0 0.01 0 277.88 0 0.32 0 97854 0 1389401 0 185826 0 11960 0 5624 0 0 0 438894 0 5 0 0 0 185 0 21724 0 439 0 22353 0 53.77 0 747097 0 6838 25132 3.675343667739 1389401.0 932923.0 97854.0 185826.0 11960.0 5624.0 0.0 438894.0 747097.0 67.1 7.0 13.4 0.9 0.4 0.0 31.6 53.8 43 43 43.00 38 59744243 26.0 22.8 22.7 28.5 0.0 36.4 25.2 smartseq 1073227 SRR2088604 SRP060416 SRS980003 SRX1082573 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811045: T86_P3_D5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811045 GSM1811045 T86_P3_D5_ILC1 56390200 1311400 2016-01-28 01:00:06 61006942 56390200 1311400 1 1311400 index:0,count:1311400,average:43,stdev:0 GSM1811045_r1 5.77 5.73 0.24 36135960 43912120 28153190 35049156 121.52 124.49 0 0 0 0 0 0 48.43 62.8 1473745 422269 1473745 422269 57.44 60.01 1473745 500762 1473745 403465 6833331 18.91 7.13 0 15.21 0 1.19 0 0.72 0 0.00 0 31.61 0 871846 0 43 0 41.87 0 1.14 0 0.01 0 1.14 0 0.01 0 262.28 0 0.32 0 93440 0 1311400 0 199491 0 15646 0 9422 0 0 0 414486 0 6 0 0 0 124 0 17446 0 361 0 17937 0 51.27 0 672355 0 6068 22306 3.676005273566 1311400.0 871846.0 93440.0 199491.0 15646.0 9422.0 0.0 414486.0 672355.0 66.5 7.1 15.2 1.2 0.7 0.0 31.6 51.3 43 43 43.00 38 56390200 26.1 22.8 22.8 28.2 0.0 36.5 25.4 smartseq 1073243 SRR2088605 SRP060416 SRS980002 SRX1082574 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811046: T86_P3_D6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811046 GSM1811046 T86_P3_D6_ILC1 70776022 1645954 2016-01-28 01:00:06 76303967 70776022 1645954 1 1645954 index:0,count:1645954,average:43,stdev:0 GSM1811046_r1 10.02 5.61 0.17 48760890 66950278 38037829 53816821 137.3 141.48 0 0 0 0 0 0 63.68 82.46 1911685 749238 1911685 749238 73.75 78.81 1911685 867728 1911685 716113 4734081 9.71 6.08 0 16.28 0 0.91 0 0.33 0 0.00 0 27.27 0 1176636 0 43 0 41.86 0 1.11 0 0.01 0 1.09 0 0.00 0 257.63 0 0.32 0 100023 0 1645954 0 267989 0 14963 0 5445 0 0 0 448910 0 16 0 0 0 252 0 30593 0 439 0 31300 0 55.20 0 908647 0 8764 37152 4.239160200822 1645954.0 1176636.0 100023.0 267989.0 14963.0 5445.0 0.0 448910.0 908647.0 71.5 6.1 16.3 0.9 0.3 0.0 27.3 55.2 43 43 43.00 38 70776022 25.8 23.0 23.0 28.1 0.0 36.5 25.3 smartseq 1073258 SRR2088606 SRP060416 SRS980001 SRX1082575 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811047: T86_P3_D7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811047 GSM1811047 T86_P3_D7_ILC1 57831345 1344915 2016-01-28 01:00:06 62622275 57831345 1344915 1 1344915 index:0,count:1344915,average:43,stdev:0 GSM1811047_r1 6.77 6.98 0.22 35850678 46218142 27664873 37028087 128.92 133.85 0 0 0 0 0 0 57.3 75.41 1475651 500749 1475651 500749 65.32 71.69 1475651 570749 1475651 476102 4885498 13.63 7.49 0 15.60 0 1.11 0 0.41 0 0.00 0 33.51 0 873836 0 43 0 41.66 0 1.15 0 0.01 0 1.12 0 0.00 0 302.61 0 0.33 0 100667 0 1344915 0 209770 0 14905 0 5511 0 0 0 450663 0 10 0 0 0 173 0 20885 0 464 0 21532 0 49.38 0 664066 0 6722 25276 3.760190419518 1344915.0 873836.0 100667.0 209770.0 14905.0 5511.0 0.0 450663.0 664066.0 65.0 7.5 15.6 1.1 0.4 0.0 33.5 49.4 43 43 43.00 38 57831345 26.0 22.5 22.4 29.1 0.0 36.3 24.8 smartseq 1073274 SRR2088607 SRP060416 SRS980000 SRX1082576 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811048: T86_P3_D8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811048 GSM1811048 T86_P3_D8_ILC1 34783130 808910 2016-01-28 01:00:06 37916985 34783130 808910 1 808910 index:0,count:808910,average:43,stdev:0 GSM1811048_r1 6.74 7.7 0.25 19780374 25351596 15355448 20620095 128.17 134.29 0 0 0 0 0 0 58.81 77.21 822579 285623 822579 285623 65.79 74.41 822579 319547 822579 275263 2684811 13.57 8.66 0 14.31 0 0.98 0 0.25 0 0.00 0 38.73 0 485711 0 43 0 41.51 0 1.11 0 0.01 0 1.12 0 0.00 0 145.60 0 0.34 0 70017 0 808910 0 115763 0 7905 0 1996 0 0 0 313298 0 6 0 0 0 103 0 11320 0 264 0 11693 0 45.73 0 369948 0 4776 12941 2.709589614740 808910.0 485711.0 70017.0 115763.0 7905.0 1996.0 0.0 313298.0 369948.0 60.0 8.7 14.3 1.0 0.2 0.0 38.7 45.7 43 43 43.00 38 34783130 26.0 22.5 22.4 29.2 0.0 36.2 24.3 smartseq 1073289 SRR2088608 SRP060416 SRS979998 SRX1082577 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811049: T86_P3_E10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811049 GSM1811049 T86_P3_E10_ILC1 58799748 1367436 2016-01-28 01:00:06 63917013 58799748 1367436 1 1367436 index:0,count:1367436,average:43,stdev:0 GSM1811049_r1 9.82 5.58 0.16 42279083 58209656 34050104 48070256 137.68 141.18 0 0 0 0 0 0 63.62 79.63 1561681 645852 1561681 645852 72.49 76.38 1561681 735935 1561681 619483 4343437 10.27 5.49 0 14.93 0 0.86 0 0.38 0 0.00 0 24.52 0 1015210 0 43 0 41.98 0 1.13 0 0.01 0 1.12 0 0.00 0 328.18 0 0.32 0 75015 0 1367436 0 204189 0 11787 0 5181 0 0 0 335258 0 5 0 0 0 215 0 25837 0 392 0 26449 0 59.31 0 811021 0 8731 31542 3.612644599702 1367436.0 1015210.0 75015.0 204189.0 11787.0 5181.0 0.0 335258.0 811021.0 74.2 5.5 14.9 0.9 0.4 0.0 24.5 59.3 43 43 43.00 38 58799748 25.9 23.1 23.2 27.8 0.0 36.3 25.3 smartseq 1073305 SRR2088609 SRP060416 SRS979999 SRX1082578 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811050: T86_P3_E11_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811050 GSM1811050 T86_P3_E11_ILC1 135080286 3141402 2016-01-28 01:00:06 145018598 135080286 3141402 1 3141402 index:0,count:3141402,average:43,stdev:0 GSM1811050_r1 8.32 6.04 0.23 93891343 124127326 74540238 101685351 132.2 136.42 0 0 0 0 0 0 56.55 72.05 3655958 1284133 3655958 1284133 64.19 68.67 3655958 1457705 3655958 1223854 14253007 15.18 5.87 0 15.55 0 0.98 0 0.56 0 0.00 0 26.17 0 2270930 0 43 0 41.82 0 1.15 0 0.01 0 1.10 0 0.01 0 257.02 0 0.32 0 184256 0 3141402 0 488586 0 30860 0 17596 0 0 0 822016 0 18 0 0 0 410 0 50686 0 1034 0 52148 0 56.74 0 1782344 0 9056 60346 6.663648409894 3141402.0 2270930.0 184256.0 488586.0 30860.0 17596.0 0.0 822016.0 1782344.0 72.3 5.9 15.6 1.0 0.6 0.0 26.2 56.7 43 43 43.00 38 135080286 26.4 22.4 22.4 28.8 0.0 36.3 25.0 smartseq 1073417 SRR2088610 SRP060416 SRS979997 SRX1082579 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811051: T86_P3_E12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811051 GSM1811051 T86_P3_E12_ILC1 156131839 3630973 2016-01-28 01:00:06 167462663 156131839 3630973 1 3630973 index:0,count:3630973,average:43,stdev:0 GSM1811051_r1 9.63 6.39 0.19 99356886 134182225 77530316 108128577 135.05 139.47 0 0 0 0 0 0 62.37 80.98 3921837 1503880 3921837 1503880 71.77 77.89 3921837 1730575 3921837 1446598 11009667 11.08 7.25 0 15.26 0 0.90 0 0.32 0 0.00 0 32.37 0 2411168 0 43 0 41.75 0 1.13 0 0.01 0 1.11 0 0.00 0 326.79 0 0.33 0 263406 0 3630973 0 553964 0 32571 0 11718 0 0 0 1175516 0 53 0 0 0 516 0 63009 0 1050 0 64628 0 51.15 0 1857204 0 9249 74589 8.064547518651 3630973.0 2411168.0 263406.0 553964.0 32571.0 11718.0 0.0 1175516.0 1857204.0 66.4 7.3 15.3 0.9 0.3 0.0 32.4 51.1 43 43 43.00 38 156131839 25.8 22.8 22.8 28.6 0.0 36.3 24.8 smartseq 1073434 SRR2088611 SRP060416 SRS979996 SRX1082580 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811052: T86_P3_E2_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811052 GSM1811052 T86_P3_E2_ILC1 50061503 1164221 2016-01-28 01:00:06 55559041 50061503 1164221 1 1164221 index:0,count:1164221,average:43,stdev:0 GSM1811052_r1 8.61 6.16 0.22 33136484 44365373 25428791 35243909 133.89 138.6 0 0 0 0 0 0 60.56 79.88 1332372 486301 1332372 486301 70.38 76.12 1332372 565229 1332372 463399 3825428 11.54 6.75 0 16.69 0 0.87 0 0.28 0 0.00 0 29.87 0 803061 0 43 0 41.77 0 1.18 0 0.01 0 1.12 0 0.00 0 47.09 0 0.37 0 78575 0 1164221 0 194253 0 10125 0 3317 0 0 0 347718 0 5 0 0 0 146 0 22067 0 384 0 22602 0 52.29 0 608808 0 7028 26326 3.745873648264 1164221.0 803061.0 78575.0 194253.0 10125.0 3317.0 0.0 347718.0 608808.0 69.0 6.7 16.7 0.9 0.3 0.0 29.9 52.3 43 43 43.00 38 50061503 26.4 22.4 22.7 28.6 0.0 35.8 24.3 smartseq 1074040 SRR2088637 SRP060416 SRS979975 SRX1082606 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811078: T86_P3_H11_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811078 GSM1811078 T86_P3_H11_ILC1 30755750 715250 2016-01-28 01:00:06 33790067 30755750 715250 1 715250 index:0,count:715250,average:43,stdev:0 GSM1811078_r1 8.74 8.03 0.22 17848160 22786416 13237393 17624104 127.67 133.14 0 0 0 0 0 0 53.93 74.25 782119 237411 782119 237411 63.39 71.65 782119 279040 782119 229099 2888984 16.19 8.36 0 16.84 0 1.01 0 0.29 0 0.00 0 37.15 0 440207 0 43 0 41.40 0 1.14 0 0.01 0 1.14 0 0.00 0 128.75 0 0.35 0 59809 0 715250 0 120474 0 7246 0 2047 0 0 0 265750 0 10 0 0 0 70 0 8542 0 275 0 8897 0 44.70 0 319733 0 4070 10402 2.555773955774 715250.0 440207.0 59809.0 120474.0 7246.0 2047.0 0.0 265750.0 319733.0 61.5 8.4 16.8 1.0 0.3 0.0 37.2 44.7 43 43 43.00 38 30755750 26.3 21.8 21.7 30.3 0.0 36.0 24.1 smartseq 1074472 SRR2088652 SRP060416 SRS979963 SRX1082621 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811093: T86_P4_B10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811093 GSM1811093 T86_P4_B10_ILC3 35326005 821535 2016-01-28 01:00:06 40389853 35326005 821535 1 821535 index:0,count:821535,average:43,stdev:0 GSM1811093_r1 7.68 7.19 0.19 24034835 30181783 18711151 24260984 125.58 129.66 0 0 0 0 0 0 55.11 72.03 949887 324025 949887 324025 62.16 68.39 949887 365450 949887 307653 3917180 16.30 6.06 0 16.81 0 1.02 0 0.43 0 0.00 0 26.98 0 587957 0 43 0 41.60 0 1.19 0 0.01 0 1.17 0 0.00 0 173.97 0 0.44 0 49772 0 821535 0 138130 0 8420 0 3512 0 0 0 221646 0 3 0 0 0 126 0 13832 0 296 0 14257 0 54.75 0 449827 0 6413 16022 2.498362700764 821535.0 587957.0 49772.0 138130.0 8420.0 3512.0 0.0 221646.0 449827.0 71.6 6.1 16.8 1.0 0.4 0.0 27.0 54.8 43 43 43.00 38 35326005 27.0 21.1 22.1 29.8 0.0 34.8 22.9 smartseq 1074489 SRR2088653 SRP060416 SRS979960 SRX1082622 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811094: T86_P4_B12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811094 GSM1811094 T86_P4_B12_ILC3 192660124 4480468 2016-01-28 01:00:06 208109376 192660124 4480468 1 4480468 index:0,count:4480468,average:43,stdev:0 GSM1811094_r1 7.02 6.14 0.18 139282336 182514715 112818550 152291316 131.04 134.99 0 0 0 0 0 0 59.98 74.97 5095730 2022469 5095730 2022469 66.32 71.35 5095730 2236342 5095730 1925018 18019813 12.94 5.23 0 15.05 0 1.03 0 0.51 0 0.00 0 23.20 0 3372091 0 43 0 41.82 0 1.20 0 0.01 0 1.11 0 0.00 0 350.65 0 0.35 0 234284 0 4480468 0 674235 0 46083 0 22852 0 0 0 1039442 0 66 0 0 0 672 0 92393 0 1510 0 94641 0 60.21 0 2697856 0 13214 108347 8.199409716967 4480468.0 3372091.0 234284.0 674235.0 46083.0 22852.0 0.0 1039442.0 2697856.0 75.3 5.2 15.0 1.0 0.5 0.0 23.2 60.2 43 43 43.00 38 192660124 26.4 22.4 22.4 28.8 0.0 36.0 24.5 smartseq 1074505 SRR2088654 SRP060416 SRS979959 SRX1082623 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811095: T86_P4_B1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811095 GSM1811095 T86_P4_B1_ILC3 211391741 4916087 2016-01-28 01:00:06 227174149 211391741 4916087 1 4916087 index:0,count:4916087,average:43,stdev:0 GSM1811095_r1 6.28 6.51 0.22 148896542 194119233 120329648 161645028 130.37 134.34 0 0 0 0 0 0 59.29 74.35 5480835 2141587 5480835 2141587 65.82 71.08 5480835 2377534 5480835 2047507 20319750 13.65 5.62 0 14.88 0 1.00 0 0.45 0 0.00 0 25.07 0 3612118 0 43 0 41.78 0 1.18 0 0.01 0 1.13 0 0.01 0 280.92 0 0.35 0 276052 0 4916087 0 731743 0 49393 0 22238 0 0 0 1232338 0 28 0 0 0 577 0 91248 0 1599 0 93452 0 58.59 0 2880375 0 9819 108872 11.087890823913 4916087.0 3612118.0 276052.0 731743.0 49393.0 22238.0 0.0 1232338.0 2880375.0 73.5 5.6 14.9 1.0 0.5 0.0 25.1 58.6 43 43 43.00 38 211391741 26.3 22.3 22.4 28.9 0.0 36.1 24.5 smartseq 1074521 SRR2088655 SRP060416 SRS979961 SRX1082624 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811096: T86_P4_B3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811096 GSM1811096 T86_P4_B3_ILC3 176862010 4113070 2016-01-28 01:00:06 190823654 176862010 4113070 1 4113070 index:0,count:4113070,average:43,stdev:0 GSM1811096_r1 4.23 7.15 0.22 123773765 157824896 101136190 132587554 127.51 131.1 0 0 0 0 0 0 55.82 69.3 4525379 1678386 4525379 1678386 60.81 65.65 4525379 1828394 4525379 1589978 20319406 16.42 5.61 0 14.22 0 1.27 0 0.59 0 0.00 0 25.04 0 3006692 0 43 0 41.76 0 1.25 0 0.01 0 1.16 0 0.01 0 379.67 0 0.35 0 230722 0 4113070 0 584673 0 52056 0 24317 0 0 0 1030005 0 34 0 0 0 462 0 75343 0 1616 0 77455 0 58.89 0 2422019 0 10920 86784 7.947252747253 4113070.0 3006692.0 230722.0 584673.0 52056.0 24317.0 0.0 1030005.0 2422019.0 73.1 5.6 14.2 1.3 0.6 0.0 25.0 58.9 43 43 43.00 38 176862010 26.5 22.1 22.1 29.3 0.0 36.1 24.5 smartseq 1074536 SRR2088656 SRP060416 SRS979958 SRX1082625 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811097: T86_P4_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811097 GSM1811097 T86_P4_B4_NK 144047764 3349948 2016-01-28 01:00:06 156067671 144047764 3349948 1 3349948 index:0,count:3349948,average:43,stdev:0 GSM1811097_r1 6.36 6.64 0.21 103912665 138307382 84344507 115382322 133.1 136.8 0 0 0 0 0 0 63.2 78.93 3804473 1595930 3804473 1595930 69.5 75.05 3804473 1754948 3804473 1517466 12481905 12.01 5.19 0 15.02 0 1.06 0 0.46 0 0.00 0 23.09 0 2525197 0 43 0 41.71 0 1.21 0 0.01 0 1.12 0 0.00 0 90.00 0 0.35 0 173858 0 3349948 0 503191 0 35559 0 15547 0 0 0 773645 0 68 0 0 0 504 0 74038 0 1075 0 75685 0 60.36 0 2022006 0 10038 85483 8.515939430165 3349948.0 2525197.0 173858.0 503191.0 35559.0 15547.0 0.0 773645.0 2022006.0 75.4 5.2 15.0 1.1 0.5 0.0 23.1 60.4 43 43 43.00 38 144047764 26.4 22.2 22.3 29.1 0.0 36.0 24.5 smartseq 1074552 SRR2088657 SRP060416 SRS979957 SRX1082626 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811098: T86_P4_B5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811098 GSM1811098 T86_P4_B5_ILC3 186377695 4334365 2016-01-28 01:00:06 200959321 186377695 4334365 1 4334365 index:0,count:4334365,average:43,stdev:0 GSM1811098_r1 8.05 6.72 0.2 128055374 168470396 103382648 139574487 131.56 135.01 0 0 0 0 0 0 61.1 76.8 4721833 1903988 4721833 1903988 67.25 73.13 4721833 2095601 4721833 1812971 16868884 13.17 5.98 0 14.70 0 0.93 0 0.47 0 0.00 0 26.70 0 3116166 0 43 0 41.70 0 1.17 0 0.01 0 1.14 0 0.01 0 260.06 0 0.35 0 259264 0 4334365 0 636998 0 40281 0 20454 0 0 0 1157464 0 26 0 0 0 626 0 80801 0 1589 0 83042 0 57.20 0 2479168 0 9935 93399 9.401006542526 4334365.0 3116166.0 259264.0 636998.0 40281.0 20454.0 0.0 1157464.0 2479168.0 71.9 6.0 14.7 0.9 0.5 0.0 26.7 57.2 43 43 43.00 38 186377695 26.4 22.2 22.2 29.3 0.0 36.0 24.4 smartseq 1074584 SRR2088659 SRP060416 SRS979956 SRX1082628 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811100: T86_P4_B7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811100 GSM1811100 T86_P4_B7_ILC3 178157299 4143193 2016-01-28 01:00:06 192120994 178157299 4143193 1 4143193 index:0,count:4143193,average:43,stdev:0 GSM1811100_r1 7.84 6.36 0.18 130150633 171115450 106481969 143381753 131.47 134.65 0 0 0 0 0 0 61.98 76.59 4658811 1948082 4658811 1948082 68.03 72.6 4658811 2138226 4658811 1846798 17194856 13.21 5.09 0 14.46 0 0.98 0 0.51 0 0.00 0 22.65 0 3142935 0 43 0 41.86 0 1.18 0 0.01 0 1.13 0 0.01 0 80.19 0 0.34 0 211056 0 4143193 0 599306 0 40662 0 21123 0 0 0 938473 0 28 0 0 0 776 0 91040 0 1531 0 93375 0 61.39 0 2543629 0 13648 104872 7.684056271981 4143193.0 3142935.0 211056.0 599306.0 40662.0 21123.0 0.0 938473.0 2543629.0 75.9 5.1 14.5 1.0 0.5 0.0 22.7 61.4 43 43 43.00 38 178157299 26.4 22.4 22.5 28.7 0.0 36.1 24.7 smartseq 1074696 SRR2088660 SRP060416 SRS979955 SRX1082629 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811101: T86_P4_B8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811101 GSM1811101 T86_P4_B8_ILC3 69457986 1615302 2016-01-28 01:00:06 78327475 69457986 1615302 1 1615302 index:0,count:1615302,average:43,stdev:0 GSM1811101_r1 7.46 6.36 0.2 49550675 64925663 40336420 54154683 131.03 134.26 0 0 0 0 0 0 60.39 75.09 1796997 723939 1796997 723939 66.61 71.28 1796997 798582 1796997 687233 6558222 13.24 5.45 0 14.53 0 0.88 0 0.45 0 0.00 0 24.45 0 1198854 0 43 0 41.84 0 1.17 0 0.01 0 1.13 0 0.00 0 207.68 0 0.40 0 88008 0 1615302 0 234751 0 14262 0 7257 0 0 0 394929 0 16 0 0 0 223 0 32596 0 550 0 33385 0 59.69 0 964103 0 10148 37052 3.651162790698 1615302.0 1198854.0 88008.0 234751.0 14262.0 7257.0 0.0 394929.0 964103.0 74.2 5.4 14.5 0.9 0.4 0.0 24.4 59.7 43 43 43.00 38 69457986 26.9 21.9 22.6 28.6 0.0 35.3 23.6 smartseq 1074713 SRR2088661 SRP060416 SRS979954 SRX1082630 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811102: T86_P4_B9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811102 GSM1811102 T86_P4_B9_NK 46373909 1078463 2016-01-28 01:00:06 50731126 46373909 1078463 1 1078463 index:0,count:1078463,average:43,stdev:0 GSM1811102_r1 6.41 5.4 0.14 35094193 47093203 28384697 38967984 134.19 137.29 0 0 0 0 0 0 62.3 77.68 1273920 525146 1273920 525146 70.35 73.76 1273920 593029 1273920 498618 4024252 11.47 4.62 0 15.48 0 1.07 0 0.50 0 0.00 0 20.27 0 842933 0 43 0 41.99 0 1.23 0 0.01 0 1.12 0 0.00 0 228.38 0 0.34 0 49821 0 1078463 0 166902 0 11534 0 5374 0 0 0 218622 0 5 0 0 0 170 0 25870 0 362 0 26407 0 62.68 0 676031 0 9576 30718 3.207811194653 1078463.0 842933.0 49821.0 166902.0 11534.0 5374.0 0.0 218622.0 676031.0 78.2 4.6 15.5 1.1 0.5 0.0 20.3 62.7 43 43 43.00 38 46373909 26.4 22.6 22.7 28.3 0.0 36.2 25.0 smartseq 1074729 SRR2088662 SRP060416 SRS979952 SRX1082631 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811103: T86_P4_C10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811103 GSM1811103 T86_P4_C10_ILC3 47880457 1113499 2016-01-28 01:00:06 53219789 47880457 1113499 1 1113499 index:0,count:1113499,average:43,stdev:0 GSM1811103_r1 9.99 6.2 0.22 34430332 45394434 27785416 37466660 131.84 134.84 0 0 0 0 0 0 60.63 76.09 1263891 505350 1263891 505350 66.82 72.1 1263891 556967 1263891 478847 4645212 13.49 5.36 0 15.21 0 0.94 0 0.46 0 0.00 0 23.74 0 833557 0 43 0 41.83 0 1.25 0 0.01 0 1.13 0 0.00 0 250.54 0 0.36 0 59669 0 1113499 0 169374 0 10447 0 5138 0 0 0 264357 0 19 0 0 0 156 0 20225 0 408 0 20808 0 59.65 0 664183 0 8032 23031 2.867405378486 1113499.0 833557.0 59669.0 169374.0 10447.0 5138.0 0.0 264357.0 664183.0 74.9 5.4 15.2 0.9 0.5 0.0 23.7 59.6 43 43 43.00 38 47880457 26.7 22.0 22.2 29.0 0.0 35.7 24.2 smartseq 1074746 SRR2088663 SRP060416 SRS979953 SRX1082632 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811104: T86_P4_C11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811104 GSM1811104 T86_P4_C11_ILC3 96146882 2235974 2016-01-28 01:00:06 104574009 96146882 2235974 1 2235974 index:0,count:2235974,average:43,stdev:0 GSM1811104_r1 6.87 6.06 0.2 70707886 92785073 57960384 77766786 131.22 134.17 0 0 0 0 0 0 63.29 78.07 2530137 1082409 2530137 1082409 68.98 73.88 2530137 1179856 2530137 1024331 8801091 12.45 4.96 0 14.48 0 0.97 0 0.44 0 0.00 0 22.10 0 1710364 0 43 0 41.80 0 1.18 0 0.01 0 1.14 0 0.00 0 321.98 0 0.34 0 110829 0 2235974 0 323851 0 21684 0 9835 0 0 0 494091 0 24 0 0 0 337 0 51799 0 809 0 52969 0 62.01 0 1386513 0 10873 59186 5.443391888163 2235974.0 1710364.0 110829.0 323851.0 21684.0 9835.0 0.0 494091.0 1386513.0 76.5 5.0 14.5 1.0 0.4 0.0 22.1 62.0 43 43 43.00 38 96146882 26.6 22.2 22.3 28.8 0.0 36.1 24.6 smartseq 1074762 SRR2088664 SRP060416 SRS979951 SRX1082633 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811105: T86_P4_C12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811105 GSM1811105 T86_P4_C12_ILC3 150410861 3497927 2016-01-28 01:00:06 163064129 150410861 3497927 1 3497927 index:0,count:3497927,average:43,stdev:0 GSM1811105_r1 6.45 6.0 0.19 110639139 143207379 90436796 119787980 129.44 132.45 0 0 0 0 0 0 60.11 74.39 3976261 1606177 3976261 1606177 66.26 70.77 3976261 1770510 3976261 1528086 16029618 14.49 4.95 0 14.67 0 1.13 0 0.59 0 0.00 0 21.88 0 2672246 0 43 0 41.89 0 1.25 0 0.01 0 1.14 0 0.00 0 419.75 0 0.34 0 173227 0 3497927 0 513089 0 39567 0 20730 0 0 0 765384 0 8 0 0 0 582 0 74501 0 1210 0 76301 0 61.73 0 2159157 0 13483 87888 6.518430616332 3497927.0 2672246.0 173227.0 513089.0 39567.0 20730.0 0.0 765384.0 2159157.0 76.4 5.0 14.7 1.1 0.6 0.0 21.9 61.7 43 43 43.00 38 150410861 26.7 22.1 22.2 29.0 0.0 36.1 24.6 smartseq 1074778 SRR2088665 SRP060416 SRS979950 SRX1082634 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811106: T86_P4_C1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811106 GSM1811106 T86_P4_C1_ILC3 193349844 4496508 2016-01-28 01:00:06 207552875 193349844 4496508 1 4496508 index:0,count:4496508,average:43,stdev:0 GSM1811106_r1 9.18 6.0 0.22 148884015 200266919 121649254 166828817 134.51 137.14 0 0 0 0 0 0 62.45 77.12 5273651 2236902 5273651 2236902 69.2 72.88 5273651 2478841 5273651 2113760 17358129 11.66 4.22 0 15.16 0 1.05 0 0.53 0 0.00 0 18.76 0 3582123 0 43 0 41.94 0 1.18 0 0.01 0 1.12 0 0.00 0 311.30 0 0.33 0 189699 0 4496508 0 681720 0 47099 0 23901 0 0 0 843385 0 27 0 0 0 768 0 100675 0 1531 0 103001 0 64.50 0 2900403 0 13598 116759 8.586483306369 4496508.0 3582123.0 189699.0 681720.0 47099.0 23901.0 0.0 843385.0 2900403.0 79.7 4.2 15.2 1.0 0.5 0.0 18.8 64.5 43 43 43.00 38 193349844 26.5 22.5 22.6 28.5 0.0 36.2 25.0 smartseq 1074794 SRR2088666 SRP060416 SRS979949 SRX1082635 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811107: T86_P4_C2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811107 GSM1811107 T86_P4_C2_ILC3 44329646 1030922 2016-01-28 01:00:06 49507559 44329646 1030922 1 1030922 index:0,count:1030922,average:43,stdev:0 GSM1811107_r1 7.88 8.73 0.23 27419752 35149459 21566808 28747118 128.19 133.29 0 0 0 0 0 0 58.01 75.29 1072910 391404 1072910 391404 63.76 71.73 1072910 430178 1072910 372935 4281956 15.62 7.42 0 15.01 0 1.02 0 0.30 0 0.00 0 33.24 0 674685 0 43 0 41.48 0 1.21 0 0.01 0 1.16 0 0.01 0 265.09 0 0.39 0 76460 0 1030922 0 154789 0 10484 0 3107 0 0 0 342646 0 7 0 0 0 126 0 15461 0 382 0 15976 0 50.43 0 519896 0 6036 17464 2.893306825712 1030922.0 674685.0 76460.0 154789.0 10484.0 3107.0 0.0 342646.0 519896.0 65.4 7.4 15.0 1.0 0.3 0.0 33.2 50.4 43 43 43.00 38 44329646 26.6 21.5 21.8 30.1 0.0 35.5 23.5 smartseq 1074810 SRR2088667 SRP060416 SRS979948 SRX1082636 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811108: T86_P4_C3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811108 GSM1811108 T86_P4_C3_ILC3 101863904 2368928 2016-01-28 01:00:06 110652320 101863904 2368928 1 2368928 index:0,count:2368928,average:43,stdev:0 GSM1811108_r1 7.54 8.06 0.23 65030015 82736348 51115823 67165572 127.23 131.4 0 0 0 0 0 0 57.66 74.7 2538028 918578 2538028 918578 64.03 71.12 2538028 1020105 2538028 874456 9913331 15.24 7.08 0 15.35 0 1.08 0 0.42 0 0.00 0 31.25 0 1593132 0 43 0 41.57 0 1.23 0 0.01 0 1.15 0 0.01 0 250.83 0 0.36 0 167827 0 2368928 0 363521 0 25576 0 9944 0 0 0 740276 0 23 0 0 0 346 0 38226 0 867 0 39462 0 51.91 0 1229611 0 8874 43963 4.954135677259 2368928.0 1593132.0 167827.0 363521.0 25576.0 9944.0 0.0 740276.0 1229611.0 67.3 7.1 15.3 1.1 0.4 0.0 31.2 51.9 43 43 43.00 38 101863904 26.5 21.8 21.7 30.1 0.0 36.0 24.2 smartseq 1074825 SRR2088668 SRP060416 SRS979947 SRX1082637 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811109: T86_P4_C4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811109 GSM1811109 T86_P4_C4_ILC3 105749470 2459290 2016-01-28 01:00:06 114981065 105749470 2459290 1 2459290 index:0,count:2459290,average:43,stdev:0 GSM1811109_r1 7.29 7.72 0.22 71646697 93656302 56714384 76346521 130.72 134.62 0 0 0 0 0 0 61.34 78.72 2696565 1072123 2696565 1072123 67.84 74.36 2696565 1185714 2696565 1012750 9373232 13.08 6.27 0 15.69 0 0.99 0 0.37 0 0.00 0 27.57 0 1747866 0 43 0 41.64 0 1.24 0 0.01 0 1.15 0 0.00 0 245.93 0 0.35 0 154239 0 2459290 0 385941 0 24434 0 9041 0 0 0 677949 0 35 0 0 0 311 0 51078 0 918 0 52342 0 55.38 0 1361925 0 11114 58881 5.297912542739 2459290.0 1747866.0 154239.0 385941.0 24434.0 9041.0 0.0 677949.0 1361925.0 71.1 6.3 15.7 1.0 0.4 0.0 27.6 55.4 43 43 43.00 38 105749470 26.3 22.0 22.0 29.7 0.0 36.0 24.3 smartseq 1074841 SRR2088669 SRP060416 SRS979946 SRX1082638 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811110: T86_P4_C6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811110 GSM1811110 T86_P4_C6_ILC3 160942550 3742850 2016-01-28 01:00:06 174130807 160942550 3742850 1 3742850 index:0,count:3742850,average:43,stdev:0 GSM1811110_r1 7.33 6.26 0.19 117977919 154928615 97191319 130847040 131.32 134.63 0 0 0 0 0 0 62.12 76.31 4203234 1774386 4203234 1774386 67.89 72.91 4203234 1939326 4203234 1695330 15299670 12.97 4.98 0 14.20 0 0.96 0 0.56 0 0.00 0 22.16 0 2856452 0 43 0 41.80 0 1.26 0 0.01 0 1.13 0 0.00 0 286.69 0 0.34 0 186410 0 3742850 0 531332 0 35848 0 21069 0 0 0 829481 0 30 0 0 0 625 0 78647 0 1241 0 80543 0 62.12 0 2325120 0 11968 90857 7.591661096257 3742850.0 2856452.0 186410.0 531332.0 35848.0 21069.0 0.0 829481.0 2325120.0 76.3 5.0 14.2 1.0 0.6 0.0 22.2 62.1 43 43 43.00 38 160942550 26.5 22.3 22.3 28.9 0.0 36.1 24.6 smartseq 1074954 SRR2088670 SRP060416 SRS979945 SRX1082639 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811111: T86_P4_C7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811111 GSM1811111 T86_P4_C7_ILC3 106050384 2466288 2016-01-28 01:00:06 115299508 106050384 2466288 1 2466288 index:0,count:2466288,average:43,stdev:0 GSM1811111_r1 6.36 6.79 0.18 75109371 99135525 61292373 82969860 131.99 135.37 0 0 0 0 0 0 64.17 79.6 2718682 1167544 2718682 1167544 70.67 76.18 2718682 1285895 2718682 1117285 8720774 11.61 5.62 0 14.31 0 0.97 0 0.38 0 0.00 0 24.87 0 1819501 0 43 0 41.79 0 1.23 0 0.01 0 1.15 0 0.01 0 56.91 0 0.34 0 138696 0 2466288 0 352821 0 23881 0 9430 0 0 0 613476 0 4 0 0 0 351 0 52172 0 932 0 53459 0 59.47 0 1466680 0 9897 60720 6.135192482570 2466288.0 1819501.0 138696.0 352821.0 23881.0 9430.0 0.0 613476.0 1466680.0 73.8 5.6 14.3 1.0 0.4 0.0 24.9 59.5 43 43 43.00 38 106050384 26.3 22.4 22.4 28.9 0.0 36.1 24.5 smartseq 1074969 SRR2088671 SRP060416 SRS979944 SRX1082640 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811112: T86_P4_C8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811112 GSM1811112 T86_P4_C8_ILC3 111845193 2601051 2016-01-28 01:00:06 121624552 111845193 2601051 1 2601051 index:0,count:2601051,average:43,stdev:0 GSM1811112_r1 8.24 5.81 0.18 84407857 112304152 69371849 94457926 133.05 136.16 0 0 0 0 0 0 63.08 77.55 2989819 1284959 2989819 1284959 69.24 73.77 2989819 1410498 2989819 1222454 10196268 12.08 4.56 0 14.61 0 0.89 0 0.49 0 0.00 0 20.30 0 2037138 0 43 0 41.86 0 1.19 0 0.01 0 1.15 0 0.01 0 292.62 0 0.34 0 118568 0 2601051 0 380094 0 23199 0 12742 0 0 0 527972 0 12 0 0 0 399 0 58626 0 872 0 59909 0 63.71 0 1657044 0 11543 68429 5.928181581911 2601051.0 2037138.0 118568.0 380094.0 23199.0 12742.0 0.0 527972.0 1657044.0 78.3 4.6 14.6 0.9 0.5 0.0 20.3 63.7 43 43 43.00 38 111845193 26.6 22.4 22.5 28.6 0.0 36.1 24.7 smartseq 1074985 SRR2088672 SRP060416 SRS979893 SRX1082641 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811113: T86_P4_C9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811113 GSM1811113 T86_P4_C9_ILC3 27848864 647648 2016-01-28 01:00:06 30512942 27848864 647648 1 647648 index:0,count:647648,average:43,stdev:0 GSM1811113_r1 7.96 6.76 0.2 19445358 25494965 15635356 21048839 131.11 134.62 0 0 0 0 0 0 60.73 76.55 717773 286289 717773 286289 67.39 72.58 717773 317678 717773 271462 2470126 12.70 5.80 0 15.04 0 1.00 0 0.44 0 0.00 0 25.77 0 471414 0 43 0 41.80 0 1.16 0 0.01 0 1.12 0 0.00 0 129.53 0 0.34 0 37587 0 647648 0 97403 0 6476 0 2834 0 0 0 166924 0 7 0 0 0 95 0 13710 0 242 0 14054 0 57.75 0 374011 0 6807 15402 2.262670780079 647648.0 471414.0 37587.0 97403.0 6476.0 2834.0 0.0 166924.0 374011.0 72.8 5.8 15.0 1.0 0.4 0.0 25.8 57.7 43 43 43.00 38 27848864 26.6 22.1 22.1 29.2 0.0 36.1 24.5 smartseq 1075001 SRR2088673 SRP060416 SRS979943 SRX1082642 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811114: T86_P4_D10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811114 GSM1811114 T86_P4_D10_ILC3 75816482 1763174 2016-01-28 01:00:06 82593716 75816482 1763174 1 1763174 index:0,count:1763174,average:43,stdev:0 GSM1811114_r1 6.91 5.83 0.2 54357951 70734215 44601874 59299423 130.13 132.95 0 0 0 0 0 0 63.59 78.4 1957184 833219 1957184 833219 69.6 74.57 1957184 912034 1957184 792583 6618614 12.18 5.38 0 14.04 0 0.95 0 0.59 0 0.00 0 24.14 0 1310313 0 43 0 41.96 0 1.18 0 0.01 0 1.15 0 0.01 0 244.13 0 0.34 0 94935 0 1763174 0 247467 0 16833 0 10393 0 0 0 425635 0 13 0 0 0 258 0 36758 0 625 0 37654 0 60.28 0 1062846 0 9311 42698 4.585758779938 1763174.0 1310313.0 94935.0 247467.0 16833.0 10393.0 0.0 425635.0 1062846.0 74.3 5.4 14.0 1.0 0.6 0.0 24.1 60.3 43 43 43.00 38 75816482 26.4 22.4 22.6 28.5 0.0 36.2 24.9 smartseq 1075018 SRR2088674 SRP060416 SRS979942 SRX1082643 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811115: T86_P4_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811115 GSM1811115 T86_P4_D11_NK 51494607 1197549 2016-01-28 01:00:06 56120005 51494607 1197549 1 1197549 index:0,count:1197549,average:43,stdev:0 GSM1811115_r1 7.16 5.98 0.24 36466289 47981187 29676970 40114009 131.58 135.17 0 0 0 0 0 0 64.52 80.26 1330890 571066 1330890 571066 70.77 76.48 1330890 626440 1330890 544223 4048891 11.10 5.55 0 14.50 0 0.92 0 0.41 0 0.00 0 24.76 0 885132 0 43 0 41.71 0 1.24 0 0.01 0 1.13 0 0.00 0 269.45 0 0.34 0 66485 0 1197549 0 173585 0 10990 0 4890 0 0 0 296537 0 6 0 0 0 200 0 27870 0 418 0 28494 0 59.42 0 711547 0 9563 31929 3.338805814075 1197549.0 885132.0 66485.0 173585.0 10990.0 4890.0 0.0 296537.0 711547.0 73.9 5.6 14.5 0.9 0.4 0.0 24.8 59.4 43 43 43.00 38 51494607 26.5 22.3 22.4 28.8 0.0 36.2 24.8 smartseq 1075035 SRR2088675 SRP060416 SRS979941 SRX1082644 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811116: T86_P4_D12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811116 GSM1811116 T86_P4_D12_ILC3 61603219 1432633 2016-01-28 01:00:06 67118015 61603219 1432633 1 1432633 index:0,count:1432633,average:43,stdev:0 GSM1811116_r1 5.81 7.29 0.21 40695373 52309325 33379522 44249279 128.54 132.56 0 0 0 0 0 0 60.71 75.24 1490703 602620 1490703 602620 65.52 71.9 1490703 650358 1490703 575874 5793459 14.24 6.60 0 13.38 0 1.01 0 0.45 0 0.00 0 29.25 0 992627 0 43 0 41.67 0 1.18 0 0.01 0 1.15 0 0.00 0 257.87 0 0.35 0 94493 0 1432633 0 191651 0 14427 0 6471 0 0 0 419108 0 14 0 0 0 191 0 26072 0 502 0 26779 0 55.91 0 800976 0 7563 29587 3.912071929129 1432633.0 992627.0 94493.0 191651.0 14427.0 6471.0 0.0 419108.0 800976.0 69.3 6.6 13.4 1.0 0.5 0.0 29.3 55.9 43 43 43.00 38 61603219 26.5 22.1 22.1 29.3 0.0 36.1 24.5 smartseq 1075050 SRR2088676 SRP060416 SRS979940 SRX1082645 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811117: T86_P4_D1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811117 GSM1811117 T86_P4_D1_ILC3 59123753 1374971 2016-01-28 01:00:06 64424636 59123753 1374971 1 1374971 index:0,count:1374971,average:43,stdev:0 GSM1811117_r1 8.78 6.19 0.2 43241797 57575635 35453203 48367708 133.15 136.43 0 0 0 0 0 0 62.92 77.65 1539744 658652 1539744 658652 69.07 74.14 1539744 723024 1539744 628869 5082409 11.75 5.04 0 14.44 0 1.00 0 0.45 0 0.00 0 22.42 0 1046762 0 43 0 41.80 0 1.15 0 0.01 0 1.14 0 0.01 0 274.99 0 0.33 0 69338 0 1374971 0 198529 0 13738 0 6246 0 0 0 308225 0 5 0 0 0 227 0 26091 0 463 0 26786 0 61.69 0 848233 0 7280 30091 4.133379120879 1374971.0 1046762.0 69338.0 198529.0 13738.0 6246.0 0.0 308225.0 848233.0 76.1 5.0 14.4 1.0 0.5 0.0 22.4 61.7 43 43 43.00 38 59123753 26.7 22.2 22.2 28.8 0.0 36.2 24.8 smartseq 1075066 SRR2088677 SRP060416 SRS979895 SRX1082646 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811118: T86_P4_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811118 GSM1811118 T86_P4_D2_NK 34893984 811488 2016-01-28 01:00:06 38173026 34893984 811488 1 811488 index:0,count:811488,average:43,stdev:0 GSM1811118_r1 7.1 5.97 0.18 26567033 35507406 22035440 30064011 133.65 136.43 0 0 0 0 0 0 62.54 76.18 929192 401319 929192 401319 67.98 72.2 929192 436232 929192 380346 3386801 12.75 4.36 0 14.16 0 1.00 0 0.48 0 0.00 0 19.44 0 641703 0 43 0 41.83 0 1.24 0 0.01 0 1.14 0 0.01 0 208.67 0 0.33 0 35405 0 811488 0 114926 0 8153 0 3894 0 0 0 157738 0 6 0 0 0 109 0 17998 0 305 0 18418 0 64.91 0 526777 0 6804 20268 2.978835978836 811488.0 641703.0 35405.0 114926.0 8153.0 3894.0 0.0 157738.0 526777.0 79.1 4.4 14.2 1.0 0.5 0.0 19.4 64.9 43 43 43.00 38 34893984 26.6 22.4 22.4 28.7 0.0 36.2 24.9 smartseq 1075083 SRR2088678 SRP060416 SRS979894 SRX1082647 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811119: T86_P4_D3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811119 GSM1811119 T86_P4_D3_ILC3 82920813 1928391 2016-01-28 01:00:06 89867736 82920813 1928391 1 1928391 index:0,count:1928391,average:43,stdev:0 GSM1811119_r1 7.93 6.36 0.21 56751302 74809486 46886481 63277955 131.82 134.96 0 0 0 0 0 0 62.49 76.61 2004530 859106 2004530 859106 67.64 72.68 2004530 929932 2004530 815057 7029935 12.39 6.25 0 13.14 0 0.81 0 0.38 0 0.00 0 27.51 0 1374810 0 43 0 41.81 0 1.23 0 0.01 0 1.13 0 0.00 0 223.94 0 0.33 0 120505 0 1928391 0 253363 0 15707 0 7288 0 0 0 530586 0 24 0 0 0 329 0 37646 0 688 0 38687 0 58.15 0 1121447 0 9837 41988 4.268374504422 1928391.0 1374810.0 120505.0 253363.0 15707.0 7288.0 0.0 530586.0 1121447.0 71.3 6.2 13.1 0.8 0.4 0.0 27.5 58.2 43 43 43.00 38 82920813 26.4 22.4 22.4 28.8 0.0 36.3 25.0 smartseq 1075099 SRR2088679 SRP060416 SRS979938 SRX1082648 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811120: T86_P4_D5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811120 GSM1811120 T86_P4_D5_ILC2 90529835 2105345 2016-01-28 01:00:06 98434265 90529835 2105345 1 2105345 index:0,count:2105345,average:43,stdev:0 GSM1811120_r1 4.92 6.79 0.25 65363315 87341587 52324741 71755580 133.62 137.14 0 0 0 0 0 0 65.04 82.21 2446123 1029443 2446123 1029443 72.71 78.06 2446123 1150791 2446123 977521 6699198 10.25 5.21 0 15.70 0 1.14 0 0.44 0 0.00 0 23.25 0 1582695 0 43 0 41.79 0 1.26 0 0.01 0 1.13 0 0.01 0 244.49 0 0.35 0 109630 0 2105345 0 330506 0 23979 0 9207 0 0 0 489464 0 3 0 0 0 320 0 44089 0 747 0 45159 0 59.48 0 1252189 0 8069 53232 6.597100012393 2105345.0 1582695.0 109630.0 330506.0 23979.0 9207.0 0.0 489464.0 1252189.0 75.2 5.2 15.7 1.1 0.4 0.0 23.2 59.5 43 43 43.00 38 90529835 26.3 22.5 22.6 28.6 0.0 36.1 24.7 smartseq 1075210 SRR2088680 SRP060416 SRS979939 SRX1082649 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811121: T86_P4_D6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811121 GSM1811121 T86_P4_D6_ILC3 66176699 1538993 2016-01-28 01:00:06 72002550 66176699 1538993 1 1538993 index:0,count:1538993,average:43,stdev:0 GSM1811121_r1 4.27 7.84 0.21 41128073 51642201 32904569 43006707 125.56 130.7 0 0 0 0 0 0 56.14 71.54 1573133 566927 1573133 566927 60.49 67.55 1573133 610881 1573133 535286 6681638 16.25 7.39 0 14.13 0 1.15 0 0.43 0 0.00 0 32.80 0 1009925 0 43 0 41.52 0 1.25 0 0.01 0 1.11 0 0.00 0 205.20 0 0.35 0 113773 0 1538993 0 217499 0 17699 0 6555 0 0 0 504814 0 10 0 0 0 161 0 23789 0 535 0 24495 0 51.49 0 792426 0 6642 26780 4.031918096959 1538993.0 1009925.0 113773.0 217499.0 17699.0 6555.0 0.0 504814.0 792426.0 65.6 7.4 14.1 1.2 0.4 0.0 32.8 51.5 43 43 43.00 38 66176699 26.2 22.0 21.9 30.0 0.0 36.1 24.3 smartseq 1075225 SRR2088681 SRP060416 SRS979937 SRX1082650 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811122: T86_P4_D7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811122 GSM1811122 T86_P4_D7_ILC3 64540850 1500950 2016-01-28 01:00:06 70276312 64540850 1500950 1 1500950 index:0,count:1500950,average:43,stdev:0 GSM1811122_r1 3.5 8.75 0.19 33426270 40201971 25965843 32505421 120.27 125.19 0 0 0 0 0 0 53.01 69.78 1369117 437831 1369117 437831 58.59 66.43 1369117 483883 1369117 416826 6343665 18.98 9.99 0 13.22 0 0.92 0 0.31 0 0.00 0 43.74 0 825937 0 43 0 41.38 0 1.22 0 0.01 0 1.11 0 0.01 0 245.61 0 0.36 0 149896 0 1500950 0 198449 0 13780 0 4702 0 0 0 656531 0 5 0 0 0 234 0 17244 0 748 0 18231 0 41.81 0 627488 0 5223 19608 3.754164273406 1500950.0 825937.0 149896.0 198449.0 13780.0 4702.0 0.0 656531.0 627488.0 55.0 10.0 13.2 0.9 0.3 0.0 43.7 41.8 43 43 43.00 38 64540850 27.3 20.9 21.3 30.5 0.0 36.1 24.3 smartseq 1075241 SRR2088682 SRP060416 SRS979936 SRX1082651 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811123: T86_P4_D8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811123 GSM1811123 T86_P4_D8_ILC3 67687676 1574132 2016-01-28 01:00:06 73796258 67687676 1574132 1 1574132 index:0,count:1574132,average:43,stdev:0 GSM1811123_r1 9.03 5.07 0.2 53344664 72491649 43259293 59802374 135.89 138.24 0 0 0 0 0 0 64.84 80.58 1907701 830869 1907701 830869 72.71 76.34 1907701 931733 1907701 787135 5496817 10.30 3.83 0 15.90 0 0.89 0 0.48 0 0.00 0 17.23 0 1281413 0 43 0 41.95 0 1.20 0 0.01 0 1.11 0 0.00 0 269.85 0 0.33 0 60352 0 1574132 0 250292 0 14035 0 7491 0 0 0 271193 0 22 0 0 0 253 0 40133 0 509 0 40917 0 65.50 0 1031121 0 12319 47340 3.842844386720 1574132.0 1281413.0 60352.0 250292.0 14035.0 7491.0 0.0 271193.0 1031121.0 81.4 3.8 15.9 0.9 0.5 0.0 17.2 65.5 43 43 43.00 38 67687676 26.4 22.7 22.8 28.1 0.0 36.2 25.1 smartseq 1075257 SRR2088683 SRP060416 SRS979935 SRX1082652 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811124: T86_P4_E10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811124 GSM1811124 T86_P4_E10_ILC3 69712804 1621228 2016-01-28 01:00:06 76199521 69712804 1621228 1 1621228 index:0,count:1621228,average:43,stdev:0 GSM1811124_r1 8.0 6.29 0.19 49237889 64209386 40243901 53811205 130.41 133.71 0 0 0 0 0 0 60.95 75.57 1780210 726524 1780210 726524 66.31 71.71 1780210 790333 1780210 689453 6790681 13.79 5.64 0 14.22 0 0.99 0 0.54 0 0.00 0 24.96 0 1191906 0 43 0 41.86 0 1.23 0 0.01 0 1.13 0 0.00 0 194.55 0 0.34 0 91377 0 1621228 0 230516 0 15998 0 8736 0 0 0 404588 0 7 0 0 0 303 0 31065 0 617 0 31992 0 59.30 0 961390 0 8838 35534 4.020592894320 1621228.0 1191906.0 91377.0 230516.0 15998.0 8736.0 0.0 404588.0 961390.0 73.5 5.6 14.2 1.0 0.5 0.0 25.0 59.3 43 43 43.00 38 69712804 26.6 22.1 22.2 29.1 0.0 36.1 24.6 smartseq 1075274 SRR2088684 SRP060416 SRS979896 SRX1082653 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811125: T86_P4_E11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811125 GSM1811125 T86_P4_E11_ILC3 130455679 3033853 2016-01-28 01:00:06 141143033 130455679 3033853 1 3033853 index:0,count:3033853,average:43,stdev:0 GSM1811125_r1 8.52 6.22 0.18 95406121 125381783 78649688 105361324 131.42 133.96 0 0 0 0 0 0 62.06 76.15 3360096 1431628 3360096 1431628 67.8 72.17 3360096 1563965 3360096 1356811 12406412 13.00 5.12 0 14.07 0 0.91 0 0.43 0 0.00 0 22.62 0 2306722 0 43 0 41.84 0 1.19 0 0.01 0 1.16 0 0.01 0 62.41 0 0.33 0 155311 0 3033853 0 426824 0 27697 0 13053 0 0 0 686381 0 34 0 0 0 453 0 62420 0 1072 0 63979 0 61.96 0 1879898 0 10913 71293 6.532850728489 3033853.0 2306722.0 155311.0 426824.0 27697.0 13053.0 0.0 686381.0 1879898.0 76.0 5.1 14.1 0.9 0.4 0.0 22.6 62.0 43 43 43.00 38 130455679 26.6 22.2 22.2 28.9 0.0 36.2 24.8 smartseq 1075291 SRR2088685 SRP060416 SRS979933 SRX1082654 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811126: T86_P4_E12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811126 GSM1811126 T86_P4_E12_ILC3 121399449 2823243 2016-01-28 01:00:06 131587310 121399449 2823243 1 2823243 index:0,count:2823243,average:43,stdev:0 GSM1811126_r1 8.16 7.32 0.24 80282604 104668771 63173069 84908437 130.38 134.41 0 0 0 0 0 0 58.09 74.99 3092459 1137803 3092459 1137803 65.09 71.21 3092459 1274979 3092459 1080555 11678967 14.55 6.55 0 15.63 0 1.01 0 0.45 0 0.00 0 29.16 0 1958688 0 43 0 41.63 0 1.15 0 0.01 0 1.10 0 0.00 0 307.99 0 0.35 0 184834 0 2823243 0 441372 0 28612 0 12621 0 0 0 823322 0 47 0 0 0 360 0 50387 0 1049 0 51843 0 53.74 0 1517316 0 10271 59048 5.749002044592 2823243.0 1958688.0 184834.0 441372.0 28612.0 12621.0 0.0 823322.0 1517316.0 69.4 6.5 15.6 1.0 0.4 0.0 29.2 53.7 43 43 43.00 38 121399449 26.3 22.1 22.0 29.6 0.0 36.1 24.3 smartseq 1075306 SRR2088686 SRP060416 SRS979934 SRX1082655 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811127: T86_P4_E1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811127 GSM1811127 T86_P4_E1_ILC3 122358177 2845539 2016-01-28 01:00:06 132612778 122358177 2845539 1 2845539 index:0,count:2845539,average:43,stdev:0 GSM1811127_r1 1.57 6.89 0.18 86117913 106098074 71805861 91052167 123.2 126.8 0 0 0 0 0 0 60.84 74.0 3093589 1272254 3093589 1272254 64.13 69.93 3093589 1341059 3093589 1202324 12498150 14.51 5.60 0 13.07 0 1.05 0 0.49 0 0.00 0 24.97 0 2091194 0 43 0 41.76 0 1.34 0 0.00 0 1.17 0 0.01 0 292.68 0 0.34 0 159255 0 2845539 0 371878 0 29858 0 13999 0 0 0 710488 0 13 0 0 0 422 0 60710 0 986 0 62131 0 60.42 0 1719316 0 10739 70560 6.570444175435 2845539.0 2091194.0 159255.0 371878.0 29858.0 13999.0 0.0 710488.0 1719316.0 73.5 5.6 13.1 1.0 0.5 0.0 25.0 60.4 43 43 43.00 38 122358177 26.6 22.1 22.2 29.1 0.0 36.1 24.5 smartseq 1075322 SRR2088687 SRP060416 SRS979932 SRX1082656 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811128: T86_P4_E2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811128 GSM1811128 T86_P4_E2_ILC3 63059242 1466494 2016-01-28 01:00:06 69753349 63059242 1466494 1 1466494 index:0,count:1466494,average:43,stdev:0 GSM1811128_r1 8.02 6.68 0.19 46076198 60397794 37330343 50031136 131.08 134.02 0 0 0 0 0 0 62.72 78.38 1671270 700411 1671270 700411 69.06 74.3 1671270 771241 1671270 664009 5732169 12.44 5.03 0 15.22 0 0.95 0 0.47 0 0.00 0 22.43 0 1116803 0 43 0 41.77 0 1.17 0 0.01 0 1.14 0 0.01 0 263.97 0 0.37 0 73734 0 1466494 0 223151 0 13859 0 6866 0 0 0 328966 0 5 0 0 0 210 0 34330 0 516 0 35061 0 60.94 0 893652 0 10363 39643 3.825436649619 1466494.0 1116803.0 73734.0 223151.0 13859.0 6866.0 0.0 328966.0 893652.0 76.2 5.0 15.2 0.9 0.5 0.0 22.4 60.9 43 43 43.00 38 63059242 26.7 22.1 22.3 28.9 0.0 35.8 24.2 smartseq 1075338 SRR2088688 SRP060416 SRS979931 SRX1082657 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811129: T86_P4_E3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811129 GSM1811129 T86_P4_E3_ILC3 169709691 3946737 2016-01-28 01:00:06 182551748 169709691 3946737 1 3946737 index:0,count:3946737,average:43,stdev:0 GSM1811129_r1 10.24 5.68 0.18 128078202 173187839 104516919 144388050 135.22 138.15 0 0 0 0 0 0 64.75 80.14 4538541 2000711 4538541 2000711 71.84 76.21 4538541 2219924 4538541 1902624 13891005 10.85 4.59 0 15.04 0 0.84 0 0.42 0 0.00 0 20.44 0 3090094 0 43 0 41.86 0 1.17 0 0.01 0 1.15 0 0.01 0 296.01 0 0.33 0 180967 0 3946737 0 593569 0 33324 0 16496 0 0 0 806823 0 34 0 0 0 640 0 90764 0 1259 0 92697 0 63.26 0 2496525 0 13662 105431 7.717098521446 3946737.0 3090094.0 180967.0 593569.0 33324.0 16496.0 0.0 806823.0 2496525.0 78.3 4.6 15.0 0.8 0.4 0.0 20.4 63.3 43 43 43.00 38 169709691 26.6 22.3 22.3 28.7 0.0 36.2 25.0 smartseq 1075354 SRR2088689 SRP060416 SRS979930 SRX1082658 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811130: T86_P4_E4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811130 GSM1811130 T86_P4_E4_ILC3 91286506 2122942 2016-01-28 01:00:06 100779247 91286506 2122942 1 2122942 index:0,count:2122942,average:43,stdev:0 GSM1811130_r1 7.34 6.48 0.18 68582391 91346052 55861728 76234756 133.19 136.47 0 0 0 0 0 0 63.79 79.24 2468908 1057524 2468908 1057524 70.08 74.83 2468908 1161709 2468908 998660 7739562 11.29 4.65 0 15.22 0 0.90 0 0.43 0 0.00 0 20.58 0 1657716 0 43 0 41.85 0 1.24 0 0.01 0 1.11 0 0.00 0 246.54 0 0.36 0 98801 0 2122942 0 323060 0 19148 0 9216 0 0 0 436862 0 17 0 0 0 290 0 48358 0 675 0 49340 0 62.87 0 1334656 0 11814 55882 4.730150668698 2122942.0 1657716.0 98801.0 323060.0 19148.0 9216.0 0.0 436862.0 1334656.0 78.1 4.7 15.2 0.9 0.4 0.0 20.6 62.9 43 43 43.00 38 91286506 26.7 22.0 22.2 29.0 0.0 35.8 24.3 smartseq 1075465 SRR2088690 SRP060416 SRS979929 SRX1082659 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811131: T86_P4_E5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811131 GSM1811131 T86_P4_E5_ILC3 150069742 3489994 2016-01-28 01:00:06 162274217 150069742 3489994 1 3489994 index:0,count:3489994,average:43,stdev:0 GSM1811131_r1 6.33 6.94 0.21 105825150 136350014 85877052 113432636 128.84 132.09 0 0 0 0 0 0 60.46 75.54 3875693 1553605 3875693 1553605 66.29 71.57 3875693 1703487 3875693 1472044 14963374 14.14 5.59 0 14.70 0 1.09 0 0.48 0 0.00 0 24.80 0 2569696 0 43 0 41.76 0 1.24 0 0.01 0 1.18 0 0.01 0 267.32 0 0.34 0 195040 0 3489994 0 513033 0 38164 0 16655 0 0 0 865479 0 28 0 0 0 657 0 65606 0 1165 0 67456 0 58.93 0 2056663 0 10244 76069 7.425712612261 3489994.0 2569696.0 195040.0 513033.0 38164.0 16655.0 0.0 865479.0 2056663.0 73.6 5.6 14.7 1.1 0.5 0.0 24.8 58.9 43 43 43.00 38 150069742 26.5 22.1 22.1 29.3 0.0 36.1 24.5 smartseq 1075481 SRR2088691 SRP060416 SRS979927 SRX1082660 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811132: T86_P4_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811132 GSM1811132 T86_P4_E6_NK 136729164 3179748 2016-01-28 01:00:06 147862772 136729164 3179748 1 3179748 index:0,count:3179748,average:43,stdev:0 GSM1811132_r1 7.42 6.24 0.19 102072758 136611345 82768055 113331214 133.84 136.93 0 0 0 0 0 0 64.59 80.55 3705696 1596614 3705696 1596614 71.42 76.3 3705696 1765534 3705696 1512434 11825312 11.59 4.68 0 15.40 0 0.92 0 0.39 0 0.00 0 20.94 0 2472006 0 43 0 41.76 0 1.20 0 0.01 0 1.13 0 0.01 0 293.52 0 0.33 0 148927 0 3179748 0 489822 0 29335 0 12542 0 0 0 665865 0 20 0 0 0 570 0 74662 0 1116 0 76368 0 62.34 0 1982184 0 10886 87061 7.997519750138 3179748.0 2472006.0 148927.0 489822.0 29335.0 12542.0 0.0 665865.0 1982184.0 77.7 4.7 15.4 0.9 0.4 0.0 20.9 62.3 43 43 43.00 38 136729164 26.5 22.3 22.3 29.0 0.0 36.1 24.7 smartseq 1075496 SRR2088692 SRP060416 SRS979928 SRX1082661 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811133: T86_P4_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811133 GSM1811133 T86_P4_E7_NK 199286166 4634562 2016-01-28 01:00:06 214283071 199286166 4634562 1 4634562 index:0,count:4634562,average:43,stdev:0 GSM1811133_r1 6.83 6.87 0.2 144100959 190264798 120441919 162693723 132.04 135.08 0 0 0 0 0 0 65.83 79.63 5017217 2280420 5017217 2280420 70.72 76.15 5017217 2449704 5017217 2180595 17595367 12.21 5.28 0 12.95 0 0.86 0 0.48 0 0.00 0 23.92 0 3463934 0 43 0 42.06 0 1.25 0 0.01 0 1.15 0 0.00 0 264.83 0 0.33 0 244612 0 4634562 0 600334 0 39710 0 22473 0 0 0 1108445 0 36 0 0 0 791 0 97565 0 1332 0 99724 0 61.79 0 2863600 0 10411 116367 11.177312457977 4634562.0 3463934.0 244612.0 600334.0 39710.0 22473.0 0.0 1108445.0 2863600.0 74.7 5.3 13.0 0.9 0.5 0.0 23.9 61.8 43 43 43.00 38 199286166 26.0 22.7 23.0 28.3 0.0 36.1 24.9 smartseq 1075512 SRR2088693 SRP060416 SRS979926 SRX1082662 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811134: T86_P4_E8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811134 GSM1811134 T86_P4_E8_ILC3 109673134 2550538 2016-01-28 01:00:06 119335285 109673134 2550538 1 2550538 index:0,count:2550538,average:43,stdev:0 GSM1811134_r1 6.92 6.29 0.19 78759897 102861180 64134994 85711596 130.6 133.64 0 0 0 0 0 0 63.19 78.45 2872189 1202112 2872189 1202112 69.12 73.83 2872189 1314895 2872189 1131219 9453032 12.00 5.42 0 14.51 0 0.89 0 0.47 0 0.00 0 24.05 0 1902317 0 43 0 41.86 0 1.21 0 0.01 0 1.16 0 0.00 0 437.24 0 0.34 0 138286 0 2550538 0 370050 0 22812 0 11968 0 0 0 613441 0 19 0 0 0 386 0 56844 0 903 0 58152 0 60.08 0 1532267 0 11704 66408 5.673957621326 2550538.0 1902317.0 138286.0 370050.0 22812.0 11968.0 0.0 613441.0 1532267.0 74.6 5.4 14.5 0.9 0.5 0.0 24.1 60.1 43 43 43.00 38 109673134 26.4 22.5 22.6 28.6 0.0 36.1 24.7 smartseq 1075528 SRR2088694 SRP060416 SRS979925 SRX1082663 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811135: T86_P4_E9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811135 GSM1811135 T86_P4_E9_ILC3 31133720 724040 2016-01-28 01:00:06 34118723 31133720 724040 1 724040 index:0,count:724040,average:43,stdev:0 GSM1811135_r1 7.47 5.93 0.18 23155975 30595399 19182574 25923311 132.13 135.14 0 0 0 0 0 0 61.13 74.6 807778 341047 807778 341047 66.97 70.92 807778 373640 807778 324230 2997873 12.95 4.77 0 13.91 0 0.99 0 0.51 0 0.00 0 21.45 0 557893 0 43 0 41.96 0 1.21 0 0.01 0 1.17 0 0.01 0 144.81 0 0.33 0 34524 0 724040 0 100696 0 7179 0 3695 0 0 0 155273 0 1 0 0 0 99 0 14713 0 262 0 15075 0 63.15 0 457197 0 7001 16733 2.390087130410 724040.0 557893.0 34524.0 100696.0 7179.0 3695.0 0.0 155273.0 457197.0 77.1 4.8 13.9 1.0 0.5 0.0 21.4 63.1 43 43 43.00 38 31133720 26.7 22.3 22.4 28.7 0.0 36.2 24.8 smartseq 1075544 SRR2088695 SRP060416 SRS979924 SRX1082664 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811136: T86_P4_F10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811136 GSM1811136 T86_P4_F10_ILC3 74590853 1734671 2016-01-28 01:00:06 81585224 74590853 1734671 1 1734671 index:0,count:1734671,average:43,stdev:0 GSM1811136_r1 7.51 5.89 0.2 54275270 71350675 44356488 59856940 131.46 134.95 0 0 0 0 0 0 61.89 76.65 1957364 811834 1957364 811834 67.49 72.5 1957364 885222 1957364 767871 7331442 13.51 5.18 0 14.56 0 0.91 0 0.50 0 0.00 0 22.98 0 1311655 0 43 0 41.88 0 1.19 0 0.01 0 1.13 0 0.01 0 215.34 0 0.35 0 89864 0 1734671 0 252498 0 15801 0 8630 0 0 0 398585 0 22 0 0 0 293 0 36641 0 560 0 37516 0 61.06 0 1059157 0 9846 42499 4.316372130815 1734671.0 1311655.0 89864.0 252498.0 15801.0 8630.0 0.0 398585.0 1059157.0 75.6 5.2 14.6 0.9 0.5 0.0 23.0 61.1 43 43 43.00 38 74590853 26.6 22.2 22.3 28.9 0.0 36.1 24.6 smartseq 1075560 SRR2088696 SRP060416 SRS979897 SRX1082665 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811137: T86_P4_F11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811137 GSM1811137 T86_P4_F11_ILC3 169298697 3937179 2016-01-28 01:00:06 182787740 169298697 3937179 1 3937179 index:0,count:3937179,average:43,stdev:0 GSM1811137_r1 6.76 5.76 0.24 121565826 154205074 100216123 130124362 126.85 129.84 0 0 0 0 0 0 58.46 71.58 4280055 1711509 4280055 1711509 63.35 67.26 4280055 1854730 4280055 1608095 17358007 14.28 5.37 0 13.63 0 1.14 0 0.75 0 0.00 0 23.75 0 2927568 0 43 0 41.92 0 1.20 0 0.01 0 1.12 0 0.01 0 301.57 0 0.35 0 211615 0 3937179 0 536672 0 44742 0 29725 0 0 0 935144 0 40 0 0 0 594 0 80977 0 1434 0 83045 0 60.73 0 2390896 0 13730 94447 6.878878368536 3937179.0 2927568.0 211615.0 536672.0 44742.0 29725.0 0.0 935144.0 2390896.0 74.4 5.4 13.6 1.1 0.8 0.0 23.8 60.7 43 43 43.00 38 169298697 26.4 22.7 22.7 28.2 0.0 36.2 24.9 smartseq 1075592 SRR2088698 SRP060416 SRS979922 SRX1082667 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811139: T86_P4_F1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811139 GSM1811139 T86_P4_F1_ILC3 163551145 3803515 2016-01-28 01:00:06 176534980 163551145 3803515 1 3803515 index:0,count:3803515,average:43,stdev:0 GSM1811139_r1 9.08 6.48 0.2 120370513 161995551 98387460 135373378 134.58 137.59 0 0 0 0 0 0 65.41 80.95 4303335 1904518 4303335 1904518 72.4 77.44 4303335 2108142 4303335 1821838 13429321 11.16 4.98 0 14.70 0 0.90 0 0.39 0 0.00 0 22.15 0 2911704 0 43 0 41.82 0 1.16 0 0.01 0 1.13 0 0.00 0 318.43 0 0.34 0 189439 0 3803515 0 558991 0 34352 0 14932 0 0 0 842527 0 40 0 0 0 593 0 84618 0 1265 0 86516 0 61.86 0 2352713 0 11376 96810 8.510021097046 3803515.0 2911704.0 189439.0 558991.0 34352.0 14932.0 0.0 842527.0 2352713.0 76.6 5.0 14.7 0.9 0.4 0.0 22.2 61.9 43 43 43.00 38 163551145 26.6 22.3 22.4 28.8 0.0 36.1 24.7 smartseq 1075608 SRR2088699 SRP060416 SRS979920 SRX1082668 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811140: T86_P4_F2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811140 GSM1811140 T86_P4_F2_ILC3 80664689 1875923 2016-01-28 01:00:06 89190375 80664689 1875923 1 1875923 index:0,count:1875923,average:43,stdev:0 GSM1811140_r1 7.11 5.62 0.18 61670589 81633890 51502087 69601796 132.37 135.14 0 0 0 0 0 0 62.02 75.02 2118840 921863 2118840 921863 66.98 70.95 2118840 995520 2118840 871950 8056651 13.06 4.30 0 13.73 0 1.00 0 0.57 0 0.00 0 19.19 0 1486381 0 43 0 41.91 0 1.25 0 0.01 0 1.16 0 0.00 0 259.74 0 0.36 0 80628 0 1875923 0 257501 0 18765 0 10720 0 0 0 360057 0 21 0 0 0 288 0 41927 0 684 0 42920 0 65.51 0 1228880 0 11113 47797 4.300998830199 1875923.0 1486381.0 80628.0 257501.0 18765.0 10720.0 0.0 360057.0 1228880.0 79.2 4.3 13.7 1.0 0.6 0.0 19.2 65.5 43 43 43.00 38 80664689 26.7 22.3 22.5 28.5 0.0 35.8 24.4 smartseq 1077289 SRR2088702 SRP060416 SRS979918 SRX1082671 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811143: T86_P4_F5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811143 GSM1811143 T86_P4_F5_ILC3 200968670 4673690 2016-01-28 01:00:06 216366612 200968670 4673690 1 4673690 index:0,count:4673690,average:43,stdev:0 GSM1811143_r1 6.69 6.02 0.18 153583184 204616230 127319501 173372452 133.23 136.17 0 0 0 0 0 0 63.93 77.86 5324879 2366063 5324879 2366063 69.45 73.66 5324879 2570457 5324879 2238411 17614765 11.47 4.36 0 14.17 0 0.94 0 0.46 0 0.00 0 19.42 0 3700968 0 43 0 41.90 0 1.24 0 0.01 0 1.17 0 0.01 0 350.53 0 0.34 0 203736 0 4673690 0 662175 0 43750 0 21420 0 0 0 907552 0 39 0 0 0 900 0 105806 0 1658 0 108403 0 65.02 0 3038793 0 13010 122965 9.451575710992 4673690.0 3700968.0 203736.0 662175.0 43750.0 21420.0 0.0 907552.0 3038793.0 79.2 4.4 14.2 0.9 0.5 0.0 19.4 65.0 43 43 43.00 38 200968670 26.4 22.6 22.7 28.4 0.0 36.1 24.8 smartseq 1077304 SRR2088703 SRP060416 SRS979917 SRX1082672 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811144: T86_P4_F6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811144 GSM1811144 T86_P4_F6_ILC3 134413184 3125888 2016-01-28 01:00:06 145917554 134413184 3125888 1 3125888 index:0,count:3125888,average:43,stdev:0 GSM1811144_r1 6.49 6.34 0.21 97835157 127914440 79138936 106039400 130.74 133.99 0 0 0 0 0 0 64.91 81.21 3580716 1539754 3580716 1539754 71.23 77.28 3580716 1689715 3580716 1465336 11162419 11.41 5.10 0 15.23 0 0.98 0 0.40 0 0.00 0 22.73 0 2372181 0 43 0 41.74 0 1.19 0 0.01 0 1.10 0 0.00 0 401.90 0 0.35 0 159567 0 3125888 0 476136 0 30645 0 12520 0 0 0 710542 0 14 0 0 0 488 0 72071 0 1129 0 73702 0 60.66 0 1896045 0 10813 83650 7.736058448164 3125888.0 2372181.0 159567.0 476136.0 30645.0 12520.0 0.0 710542.0 1896045.0 75.9 5.1 15.2 1.0 0.4 0.0 22.7 60.7 43 43 43.00 38 134413184 26.6 22.2 22.2 29.1 0.0 36.1 24.6 smartseq 1077320 SRR2088704 SRP060416 SRS979916 SRX1082673 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811145: T86_P4_F7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811145 GSM1811145 T86_P4_F7_ILC3 166813512 3879384 2016-01-28 01:00:06 180222547 166813512 3879384 1 3879384 index:0,count:3879384,average:43,stdev:0 GSM1811145_r1 7.71 5.71 0.18 129647811 174742415 109113820 149800548 134.78 137.29 0 0 0 0 0 0 65.87 78.91 4385672 2052620 4385672 2052620 71.17 74.75 4385672 2217740 4385672 1944631 13837256 10.67 4.12 0 13.27 0 0.86 0 0.46 0 0.00 0 18.34 0 3116303 0 43 0 41.95 0 1.19 0 0.01 0 1.13 0 0.00 0 410.76 0 0.34 0 159689 0 3879384 0 514961 0 33464 0 17950 0 0 0 711667 0 29 0 0 0 620 0 91126 0 1426 0 93201 0 67.06 0 2601342 0 13883 104514 7.528199956782 3879384.0 3116303.0 159689.0 514961.0 33464.0 17950.0 0.0 711667.0 2601342.0 80.3 4.1 13.3 0.9 0.5 0.0 18.3 67.1 43 43 43.00 38 166813512 26.3 22.7 22.8 28.1 0.0 36.1 24.9 smartseq 1077336 SRR2088705 SRP060416 SRS979914 SRX1082674 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811146: T86_P4_F8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811146 GSM1811146 T86_P4_F8_ILC3 111750034 2598838 2016-01-28 01:00:06 121718334 111750034 2598838 1 2598838 index:0,count:2598838,average:43,stdev:0 GSM1811146_r1 8.6 5.82 0.16 81293662 108494736 66959134 91520022 133.46 136.68 0 0 0 0 0 0 64.47 79.08 2878632 1264698 2878632 1264698 70.31 75.09 2878632 1379321 2878632 1200960 9576819 11.78 5.23 0 13.95 0 0.96 0 0.51 0 0.00 0 23.04 0 1961777 0 43 0 41.87 0 1.17 0 0.01 0 1.15 0 0.01 0 301.80 0 0.35 0 135944 0 2598838 0 362492 0 24940 0 13329 0 0 0 598792 0 27 0 0 0 437 0 54047 0 862 0 55373 0 61.54 0 1599285 0 11099 62739 5.652671411839 2598838.0 1961777.0 135944.0 362492.0 24940.0 13329.0 0.0 598792.0 1599285.0 75.5 5.2 13.9 1.0 0.5 0.0 23.0 61.5 43 43 43.00 38 111750034 26.6 22.4 22.5 28.5 0.0 36.1 24.7 smartseq 1077352 SRR2088706 SRP060416 SRS979900 SRX1082675 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811147: T86_P4_F9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811147 GSM1811147 T86_P4_F9_ILC3 30158738 701366 2016-01-28 01:00:06 33102160 30158738 701366 1 701366 index:0,count:701366,average:43,stdev:0 GSM1811147_r1 7.03 6.36 0.19 21407310 27567022 17323275 22856474 128.77 131.94 0 0 0 0 0 0 59.05 73.93 788870 305976 788870 305976 65.19 70.12 788870 337811 788870 290204 3153297 14.73 5.49 0 14.87 0 0.95 0 0.43 0 0.00 0 24.73 0 518184 0 43 0 41.86 0 1.18 0 0.01 0 1.17 0 0.01 0 229.54 0 0.35 0 38522 0 701366 0 104319 0 6689 0 3021 0 0 0 173472 0 13 0 0 0 93 0 13399 0 240 0 13745 0 59.01 0 413865 0 6029 15266 2.532094874772 701366.0 518184.0 38522.0 104319.0 6689.0 3021.0 0.0 173472.0 413865.0 73.9 5.5 14.9 1.0 0.4 0.0 24.7 59.0 43 43 43.00 38 30158738 26.8 22.0 22.1 29.1 0.0 36.1 24.6 smartseq 1077368 SRR2088707 SRP060416 SRS979915 SRX1082676 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811148: T86_P4_G10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811148 GSM1811148 T86_P4_G10_ILC3 71569931 1664417 2016-01-28 01:00:06 78107945 71569931 1664417 1 1664417 index:0,count:1664417,average:43,stdev:0 GSM1811148_r1 6.49 6.48 0.19 52114790 67945101 42738605 57106141 130.38 133.62 0 0 0 0 0 0 61.93 76.4 1882572 779478 1882572 779478 68.1 72.88 1882572 857218 1882572 743572 6685078 12.83 5.17 0 14.33 0 0.97 0 0.52 0 0.00 0 22.88 0 1258742 0 43 0 41.89 0 1.20 0 0.01 0 1.12 0 0.00 0 249.66 0 0.34 0 86054 0 1664417 0 238498 0 16136 0 8697 0 0 0 380842 0 22 0 0 0 231 0 34644 0 609 0 35506 0 61.30 0 1020244 0 9438 40076 4.246238609875 1664417.0 1258742.0 86054.0 238498.0 16136.0 8697.0 0.0 380842.0 1020244.0 75.6 5.2 14.3 1.0 0.5 0.0 22.9 61.3 43 43 43.00 38 71569931 26.6 22.2 22.3 28.8 0.0 36.1 24.7 smartseq 1077384 SRR2088708 SRP060416 SRS979898 SRX1082677 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811149: T86_P4_G12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811149 GSM1811149 T86_P4_G12_ILC3 183270042 4262094 2016-01-28 01:00:06 196445024 183270042 4262094 1 4262094 index:0,count:4262094,average:43,stdev:0 GSM1811149_r1 8.01 4.94 0.18 145335249 194636656 119177312 162505268 133.92 136.36 0 0 0 0 0 0 63.46 78.01 5116989 2215867 5116989 2215867 70.68 73.88 5116989 2467682 5116989 2098369 16946048 11.66 3.73 0 15.28 0 0.92 0 0.50 0 0.00 0 16.66 0 3491572 0 43 0 41.96 0 1.21 0 0.01 0 1.15 0 0.00 0 356.83 0 0.32 0 159168 0 4262094 0 651254 0 39180 0 21398 0 0 0 709944 0 47 0 0 0 687 0 104306 0 1537 0 106577 0 66.64 0 2840318 0 15316 123555 8.067054061113 4262094.0 3491572.0 159168.0 651254.0 39180.0 21398.0 0.0 709944.0 2840318.0 81.9 3.7 15.3 0.9 0.5 0.0 16.7 66.6 43 43 43.00 38 183270042 26.6 22.5 22.5 28.4 0.0 36.3 25.2 smartseq 1077400 SRR2088709 SRP060416 SRS979912 SRX1082678 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811150: T86_P4_G1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811150 GSM1811150 T86_P4_G1_ILC3 171707901 3993207 2016-01-28 01:00:06 184848905 171707901 3993207 1 3993207 index:0,count:3993207,average:43,stdev:0 GSM1811150_r1 6.75 6.27 0.24 125640375 166066121 101705239 138361133 132.18 136.04 0 0 0 0 0 0 59.65 74.57 4567784 1814832 4567784 1814832 65.8 70.83 4567784 2001814 4567784 1723839 16294559 12.97 4.91 0 15.24 0 1.49 0 0.54 0 0.00 0 21.78 0 3042284 0 43 0 41.79 0 1.23 0 0.01 0 1.13 0 0.01 0 299.49 0 0.35 0 195994 0 3993207 0 608562 0 59667 0 21426 0 0 0 869830 0 41 0 0 0 641 0 77029 0 1437 0 79148 0 60.95 0 2433722 0 10306 90356 8.767320007762 3993207.0 3042284.0 195994.0 608562.0 59667.0 21426.0 0.0 869830.0 2433722.0 76.2 4.9 15.2 1.5 0.5 0.0 21.8 60.9 43 43 43.00 38 171707901 26.4 22.4 22.5 28.7 0.0 36.1 24.6 smartseq 1077513 SRR2088710 SRP060416 SRS979913 SRX1082679 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811151: T86_P4_G3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811151 GSM1811151 T86_P4_G3_ILC1 132905346 3090822 2016-01-28 01:00:06 143386616 132905346 3090822 1 3090822 index:0,count:3090822,average:43,stdev:0 GSM1811151_r1 3.9 6.81 0.2 88936230 113077047 70408399 92837420 127.14 131.86 0 0 0 0 0 0 57.89 74.32 3526801 1257224 3526801 1257224 64.27 70.77 3526801 1395607 3526801 1197149 12753824 14.34 6.31 0 15.53 0 1.23 0 0.43 0 0.00 0 28.08 0 2171612 0 43 0 41.62 0 1.24 0 0.01 0 1.12 0 0.01 0 309.08 0 0.35 0 195017 0 3090822 0 479906 0 37936 0 13443 0 0 0 867831 0 13 0 0 0 416 0 46858 0 1056 0 48343 0 54.73 0 1691706 0 7856 57349 7.300025458248 3090822.0 2171612.0 195017.0 479906.0 37936.0 13443.0 0.0 867831.0 1691706.0 70.3 6.3 15.5 1.2 0.4 0.0 28.1 54.7 43 43 43.00 38 132905346 26.7 21.9 22.0 29.5 0.0 36.1 24.5 smartseq 1077528 SRR2088711 SRP060416 SRS979911 SRX1082680 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811152: T86_P4_G5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811152 GSM1811152 T86_P4_G5_ILC3 162273529 3773803 2016-01-28 01:00:06 175131680 162273529 3773803 1 3773803 index:0,count:3773803,average:43,stdev:0 GSM1811152_r1 7.4 8.33 0.24 97650045 122532978 74230233 96641602 125.48 130.19 0 0 0 0 0 0 56.45 75.88 4019529 1359976 4019529 1359976 63.55 72.12 4019529 1531231 4019529 1292553 15678876 16.06 7.82 0 16.35 0 1.09 0 0.32 0 0.00 0 34.74 0 2409373 0 43 0 41.42 0 1.19 0 0.01 0 1.10 0 0.00 0 277.26 0 0.37 0 295191 0 3773803 0 617114 0 41292 0 12023 0 0 0 1311115 0 32 0 0 0 496 0 58195 0 1409 0 60132 0 47.49 0 1792259 0 8266 68179 8.248124848778 3773803.0 2409373.0 295191.0 617114.0 41292.0 12023.0 0.0 1311115.0 1792259.0 63.8 7.8 16.4 1.1 0.3 0.0 34.7 47.5 43 43 43.00 38 162273529 26.2 21.6 21.7 30.5 0.0 35.9 24.0 smartseq 1077544 SRR2088712 SRP060416 SRS979899 SRX1082681 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811153: T86_P4_G6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811153 GSM1811153 T86_P4_G6_ILC3 158112505 3677035 2016-01-28 01:00:06 171131676 158112505 3677035 1 3677035 index:0,count:3677035,average:43,stdev:0 GSM1811153_r1 9.91 6.41 0.24 103786095 136099197 83033498 111165044 131.13 133.88 0 0 0 0 0 0 59.29 74.99 3857178 1491015 3857178 1491015 66.16 70.87 3857178 1664023 3857178 1409054 14079196 13.57 6.87 0 14.32 0 0.86 0 0.51 0 0.00 0 30.23 0 2514964 0 43 0 41.76 0 1.16 0 0.01 0 1.15 0 0.01 0 270.15 0 0.35 0 252795 0 3677035 0 526682 0 31655 0 18868 0 0 0 1111548 0 48 0 0 0 557 0 63607 0 1355 0 65567 0 54.07 0 1988282 0 11433 73676 6.444152890755 3677035.0 2514964.0 252795.0 526682.0 31655.0 18868.0 0.0 1111548.0 1988282.0 68.4 6.9 14.3 0.9 0.5 0.0 30.2 54.1 43 43 43.00 38 158112505 27.0 21.9 22.0 29.1 0.0 36.1 24.8 smartseq 1077768 SRR2088720 SRP060416 SRS979902 SRX1082689 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811161: T86_P4_H4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811161 GSM1811161 T86_P4_H4_ILC3 75479018 1755326 2016-01-28 01:00:06 82646662 75479018 1755326 1 1755326 index:0,count:1755326,average:43,stdev:0 GSM1811161_r1 6.44 6.57 0.25 56159649 73645703 46135390 61891657 131.14 134.15 0 0 0 0 0 0 62.72 77.3 2015708 854909 2015708 854909 68.78 73.86 2015708 937528 2015708 816818 7015804 12.49 4.66 0 14.65 0 1.02 0 0.55 0 0.00 0 20.77 0 1363088 0 43 0 41.71 0 1.22 0 0.01 0 1.18 0 0.01 0 234.04 0 0.35 0 81822 0 1755326 0 257119 0 17881 0 9733 0 0 0 364624 0 9 0 0 0 307 0 31111 0 654 0 32081 0 63.01 0 1105969 0 6384 36380 5.698621553885 1755326.0 1363088.0 81822.0 257119.0 17881.0 9733.0 0.0 364624.0 1105969.0 77.7 4.7 14.6 1.0 0.6 0.0 20.8 63.0 43 43 43.00 38 75479018 26.8 22.1 22.0 29.1 0.0 36.0 24.5 smartseq 1077785 SRR2088721 SRP060416 SRS979901 SRX1082690 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811162: T86_P4_H5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811162 GSM1811162 T86_P4_H5_ILC3 128185408 2981056 2016-01-28 01:00:06 139472347 128185408 2981056 1 2981056 index:0,count:2981056,average:43,stdev:0 GSM1811162_r1 3.66 6.87 0.21 88769632 111072863 72646615 93426693 125.12 128.6 0 0 0 0 0 0 60.72 75.3 3285307 1313228 3285307 1313228 65.38 71.21 3285307 1413949 3285307 1241845 11976210 13.49 5.81 0 14.04 0 1.03 0 0.47 0 0.00 0 25.96 0 2162609 0 43 0 41.66 0 1.22 0 0.01 0 1.14 0 0.01 0 275.17 0 0.36 0 173221 0 2981056 0 418657 0 30652 0 13888 0 0 0 773907 0 36 0 0 0 345 0 58280 0 1117 0 59778 0 58.50 0 1743952 0 7782 67346 8.654073502956 2981056.0 2162609.0 173221.0 418657.0 30652.0 13888.0 0.0 773907.0 1743952.0 72.5 5.8 14.0 1.0 0.5 0.0 26.0 58.5 43 43 43.00 38 128185408 26.6 22.1 22.2 29.1 0.0 35.9 24.2 smartseq 1077800 SRR2088722 SRP060416 SRS979905 SRX1082691 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811163: T86_P4_H8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811163 GSM1811163 T86_P4_H8_ILC3 73432949 1707743 2016-01-28 01:00:06 80733896 73432949 1707743 1 1707743 index:0,count:1707743,average:43,stdev:0 GSM1811163_r1 8.94 7.98 0.31 45208009 58241066 34277205 45678479 128.83 133.26 0 0 0 0 0 0 54.84 73.71 1836932 608772 1836932 608772 62.57 70.19 1836932 694630 1836932 579725 7238540 16.01 7.62 0 16.65 0 1.08 0 0.43 0 0.00 0 33.49 0 1110181 0 43 0 41.50 0 1.20 0 0.01 0 1.16 0 0.00 0 192.12 0 0.38 0 130118 0 1707743 0 284299 0 18391 0 7318 0 0 0 571853 0 13 0 0 0 219 0 24692 0 638 0 25562 0 48.36 0 825882 0 6793 28592 4.209038716326 1707743.0 1110181.0 130118.0 284299.0 18391.0 7318.0 0.0 571853.0 825882.0 65.0 7.6 16.6 1.1 0.4 0.0 33.5 48.4 43 43 43.00 38 73432949 26.4 21.6 21.7 30.3 0.0 35.8 23.9 smartseq 526919 SRR2088148 SRP060416 SRS980341 SRX1082117 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810583: T74_P4_H8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T74 GEO Accession;;GSM1810583 GSM1810583 T74_P4_H8_ILC3 146689383 3411381 2016-01-28 01:00:06 158019236 146689383 3411381 1 3411381 index:0,count:3411381,average:43,stdev:0 GSM1810583_r1 2.55 5.85 0.23 99843440 123413602 78518262 102251018 123.61 130.23 0 0 0 0 0 0 58.45 75.22 4232691 1409517 4232691 1409517 64.41 71.21 4232691 1553255 4232691 1334329 13676152 13.70 5.72 0 15.76 0 0.91 0 0.39 0 0.00 0 28.02 0 2411409 0 43 0 41.90 0 1.42 0 0.00 0 1.19 0 0.00 0 240.80 0 0.30 0 195172 0 3411381 0 537667 0 31022 0 13204 0 0 0 955746 0 9 0 0 0 772 0 70039 0 922 0 71742 0 54.93 0 1873742 0 8035 81861 10.188052271313 3411381.0 2411409.0 195172.0 537667.0 31022.0 13204.0 0.0 955746.0 1873742.0 70.7 5.7 15.8 0.9 0.4 0.0 28.0 54.9 43 43 43.00 38 146689383 26.1 22.4 22.4 29.2 0.0 36.2 24.8 smartseq 527255 SRR2088172 SRP060416 SRS980318 SRX1082141 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810607: T75_P1_C5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810607 GSM1810607 T75_P1_C5_ILC3 107508557 2500199 2016-01-28 01:00:06 119193115 107508557 2500199 1 2500199 index:0,count:2500199,average:43,stdev:0 GSM1810607_r1 1.74 7.28 0.16 67710977 79960501 53638177 66506944 118.09 123.99 0 0 0 0 0 0 56.58 72.81 2796685 938587 2796685 938587 60.75 69.18 2796685 1007661 2796685 891857 11193856 16.53 6.47 0 14.79 0 1.09 0 0.39 0 0.00 0 32.18 0 1658803 0 43 0 41.61 0 1.30 0 0.00 0 1.16 0 0.00 0 264.73 0 0.32 0 161660 0 2500199 0 369698 0 27148 0 9700 0 0 0 804548 0 31 0 0 0 447 0 44786 0 989 0 46253 0 51.56 0 1289105 0 5254 51036 9.713741910925 2500199.0 1658803.0 161660.0 369698.0 27148.0 9700.0 0.0 804548.0 1289105.0 66.3 6.5 14.8 1.1 0.4 0.0 32.2 51.6 43 43 43.00 38 107508557 26.4 21.5 21.5 30.6 0.0 35.7 24.2 smartseq 527263 SRR2088173 SRP060416 SRS980319 SRX1082142 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810608: T75_P1_C6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810608 GSM1810608 T75_P1_C6_ILC3 136524011 3174977 2016-01-28 01:00:06 150003012 136524011 3174977 1 3174977 index:0,count:3174977,average:43,stdev:0 GSM1810608_r1 5.14 6.67 0.2 92632790 117135955 72311460 95777243 126.45 132.45 0 0 0 0 0 0 58.85 76.6 3866486 1327157 3866486 1327157 64.74 72.57 3866486 1460022 3866486 1257337 12944615 13.97 5.56 0 16.46 0 0.96 0 0.37 0 0.00 0 27.64 0 2255253 0 43 0 41.74 0 1.31 0 0.00 0 1.16 0 0.00 0 243.19 0 0.31 0 176522 0 3174977 0 522627 0 30535 0 11653 0 0 0 877536 0 15 0 0 0 617 0 57923 0 1074 0 59629 0 54.57 0 1732626 0 4966 68491 13.791985501410 3174977.0 2255253.0 176522.0 522627.0 30535.0 11653.0 0.0 877536.0 1732626.0 71.0 5.6 16.5 1.0 0.4 0.0 27.6 54.6 43 43 43.00 38 136524011 26.6 21.6 21.6 30.3 0.0 35.8 24.5 smartseq 527271 SRR2088174 SRP060416 SRS980317 SRX1082143 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810609: T75_P1_C7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810609 GSM1810609 T75_P1_C7_ILC3 88648413 2061591 2016-01-28 01:00:06 98007337 88648413 2061591 1 2061591 index:0,count:2061591,average:43,stdev:0 GSM1810609_r1 4.01 8.75 0.23 52741702 63848120 38802189 49674627 121.06 128.02 0 0 0 0 0 0 53.56 74.54 2354583 701755 2354583 701755 60.44 70.96 2354583 791828 2354583 668052 9117379 17.29 6.92 0 17.89 0 1.19 0 0.38 0 0.00 0 34.87 0 1310194 0 43 0 41.22 0 1.32 0 0.00 0 1.16 0 0.00 0 212.05 0 0.34 0 142677 0 2061591 0 368778 0 24587 0 7927 0 0 0 718883 0 1 0 0 0 314 0 31071 0 1145 0 32531 0 45.66 0 941416 0 2033 35672 17.546483030005 2061591.0 1310194.0 142677.0 368778.0 24587.0 7927.0 0.0 718883.0 941416.0 63.6 6.9 17.9 1.2 0.4 0.0 34.9 45.7 43 43 43.00 38 88648413 27.0 20.9 20.7 31.4 0.0 35.7 23.9 smartseq 527279 SRR2088175 SRP060416 SRS980315 SRX1082144 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810610: T75_P1_C8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810610 GSM1810610 T75_P1_C8_ILC3 58781086 1367002 2016-01-28 01:00:06 65289274 58781086 1367002 1 1367002 index:0,count:1367002,average:43,stdev:0 GSM1810610_r1 3.65 7.31 0.17 37854067 45176930 29646017 36917029 119.34 124.53 0 0 0 0 0 0 55.85 72.77 1556079 520267 1556079 520267 61.33 70.08 1556079 571341 1556079 500992 6521633 17.23 6.03 0 15.85 0 1.24 0 0.47 0 0.00 0 30.16 0 931524 0 43 0 41.47 0 1.39 0 0.00 0 1.18 0 0.00 0 259.01 0 0.33 0 82485 0 1367002 0 216610 0 16895 0 6361 0 0 0 412222 0 2 0 0 0 246 0 21106 0 568 0 21922 0 52.30 0 714914 0 2093 24400 11.657907310081 1367002.0 931524.0 82485.0 216610.0 16895.0 6361.0 0.0 412222.0 714914.0 68.1 6.0 15.8 1.2 0.5 0.0 30.2 52.3 43 43 43.00 38 58781086 27.1 21.1 21.0 30.9 0.0 35.8 24.2 smartseq 527287 SRR2088176 SRP060416 SRS980316 SRX1082145 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810611: T75_P1_D10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810611 GSM1810611 T75_P1_D10_ILC3 37077180 862260 2016-01-28 01:00:06 41505512 37077180 862260 1 862260 index:0,count:862260,average:43,stdev:0 GSM1810611_r1 4.82 7.68 0.24 22971182 28076970 18075626 22959659 122.23 127.02 0 0 0 0 0 0 55.75 72.25 909763 314582 909763 314582 61.03 68.97 909763 344392 909763 300291 4153861 18.08 6.60 0 14.95 0 1.06 0 0.40 0 0.00 0 33.10 0 564279 0 43 0 41.51 0 1.45 0 0.00 0 1.17 0 0.00 0 206.94 0 0.33 0 56886 0 862260 0 128865 0 9110 0 3492 0 0 0 285379 0 1 0 0 0 143 0 12687 0 369 0 13200 0 50.50 0 435414 0 2263 14071 6.217852408308 862260.0 564279.0 56886.0 128865.0 9110.0 3492.0 0.0 285379.0 435414.0 65.4 6.6 14.9 1.1 0.4 0.0 33.1 50.5 43 43 43.00 38 37077180 26.9 21.3 21.1 30.7 0.0 35.7 24.1 smartseq 527295 SRR2088177 SRP060416 SRS980314 SRX1082146 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810612: T75_P1_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810612 GSM1810612 T75_P1_D11_NK 33656186 782702 2016-01-28 01:00:06 37611466 33656186 782702 1 782702 index:0,count:782702,average:43,stdev:0 GSM1810612_r1 2.72 9.44 0.29 18267742 21754104 13697543 17231291 119.08 125.8 0 0 0 0 0 0 53.11 72.78 800397 242486 800397 242486 58.12 69.38 800397 265377 800397 231142 3365297 18.42 8.04 0 15.77 0 1.23 0 0.26 0 0.00 0 40.17 0 456599 0 43 0 41.11 0 1.44 0 0.00 0 1.14 0 0.00 0 140.89 0 0.35 0 62956 0 782702 0 123441 0 9656 0 2015 0 0 0 314432 0 1 0 0 0 89 0 9494 0 352 0 9936 0 42.57 0 333158 0 1838 10732 5.838955386289 782702.0 456599.0 62956.0 123441.0 9656.0 2015.0 0.0 314432.0 333158.0 58.3 8.0 15.8 1.2 0.3 0.0 40.2 42.6 43 43 43.00 38 33656186 26.7 21.0 20.6 31.8 0.0 35.6 23.6 smartseq 527303 SRR2088178 SRP060416 SRS980313 SRX1082147 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810613: T75_P1_D12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810613 GSM1810613 T75_P1_D12_ILC3 46708578 1086246 2016-01-28 01:00:06 52110202 46708578 1086246 1 1086246 index:0,count:1086246,average:43,stdev:0 GSM1810613_r1 2.64 7.63 0.37 29376749 35636472 23563945 30037418 121.31 127.47 0 0 0 0 0 0 55.65 70.82 1165838 402371 1165838 402371 59.27 67.96 1165838 428517 1165838 386131 4971232 16.92 6.38 0 14.26 0 1.07 0 0.39 0 0.00 0 31.98 0 723052 0 43 0 41.47 0 1.37 0 0.00 0 1.23 0 0.00 0 260.70 0 0.34 0 69263 0 1086246 0 154902 0 11577 0 4220 0 0 0 347397 0 18 0 0 0 135 0 14935 0 511 0 15599 0 52.30 0 568150 0 2018 17347 8.596134786918 1086246.0 723052.0 69263.0 154902.0 11577.0 4220.0 0.0 347397.0 568150.0 66.6 6.4 14.3 1.1 0.4 0.0 32.0 52.3 43 43 43.00 38 46708578 27.2 21.1 20.8 31.0 0.0 35.7 24.0 smartseq 528655 SRR2088221 SRP060416 SRS980271 SRX1082190 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810656: T75_P2_A5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810656 GSM1810656 T75_P2_A5_ILC3 75859181 1764167 2016-01-28 01:00:06 86439874 75859181 1764167 1 1764167 index:0,count:1764167,average:43,stdev:0 GSM1810656_r1 3.0 7.12 0.24 48750033 58809826 37509303 47896456 120.64 127.69 0 0 0 0 0 0 53.68 71.0 2126650 638582 2126650 638582 58.65 66.97 2126650 697712 2126650 602321 8068718 16.55 6.33 0 16.45 0 1.12 0 0.38 0 0.00 0 31.06 0 1189575 0 43 0 41.71 0 1.37 0 0.00 0 1.18 0 0.00 0 244.27 0 0.35 0 111645 0 1764167 0 290209 0 19803 0 6776 0 0 0 548013 0 12 0 0 0 322 0 28895 0 614 0 29843 0 50.98 0 899366 0 5274 33701 6.390026545317 1764167.0 1189575.0 111645.0 290209.0 19803.0 6776.0 0.0 548013.0 899366.0 67.4 6.3 16.5 1.1 0.4 0.0 31.1 51.0 43 43 43.00 38 75859181 26.3 21.5 21.6 30.5 0.0 35.1 23.3 smartseq 528662 SRR2088222 SRP060416 SRS980272 SRX1082191 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810657: T75_P2_A7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810657 GSM1810657 T75_P2_A7_ILC3 25796560 599920 2016-01-28 01:00:06 30390029 25796560 599920 1 599920 index:0,count:599920,average:43,stdev:0 GSM1810657_r1 3.13 9.27 0.24 17033222 20358096 12777882 16188819 119.52 126.69 0 0 0 0 0 0 50.97 69.56 743460 215581 743460 215581 56.11 65.74 743460 237315 743460 203727 3011953 17.68 5.97 0 18.84 0 1.23 0 0.43 0 0.00 0 27.84 0 422937 0 43 0 41.23 0 1.27 0 0.00 0 1.22 0 0.00 0 134.98 0 0.44 0 35788 0 599920 0 113031 0 7403 0 2578 0 0 0 167002 0 1 0 0 0 132 0 10083 0 248 0 10464 0 51.66 0 309906 0 3107 11484 3.696169938848 599920.0 422937.0 35788.0 113031.0 7403.0 2578.0 0.0 167002.0 309906.0 70.5 6.0 18.8 1.2 0.4 0.0 27.8 51.7 43 43 43.00 38 25796560 26.4 19.8 21.0 32.7 0.0 33.9 21.7 smartseq 528671 SRR2088223 SRP060416 SRS980270 SRX1082192 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810658: T75_P2_A8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810658 GSM1810658 T75_P2_A8_ILC3 36817804 856228 2016-01-28 01:00:06 42197324 36817804 856228 1 856228 index:0,count:856228,average:43,stdev:0 GSM1810658_r1 4.32 5.27 0.26 29728073 37556125 23990546 31361636 126.33 130.72 0 0 0 0 0 0 58.5 73.24 1141908 419124 1141908 419124 65.49 70.59 1141908 469159 1141908 403917 4454321 14.98 3.14 0 16.84 0 1.10 0 0.51 0 0.00 0 14.73 0 716409 0 43 0 41.93 0 1.26 0 0.00 0 1.16 0 0.00 0 256.87 0 0.35 0 26862 0 856228 0 144185 0 9396 0 4332 0 0 0 126091 0 13 0 0 0 168 0 16565 0 236 0 16982 0 66.83 0 572224 0 2920 19411 6.647602739726 856228.0 716409.0 26862.0 144185.0 9396.0 4332.0 0.0 126091.0 572224.0 83.7 3.1 16.8 1.1 0.5 0.0 14.7 66.8 43 43 43.00 38 36817804 27.8 21.2 21.5 29.5 0.0 35.2 23.7 smartseq 528678 SRR2088224 SRP060416 SRS980269 SRX1082193 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810659: T75_P2_A9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810659 GSM1810659 T75_P2_A9_ILC3 18572517 431919 2016-01-28 01:00:06 21046547 18572517 431919 1 431919 index:0,count:431919,average:43,stdev:0 GSM1810659_r1 4.25 6.29 0.21 12772023 15909631 10086353 13113087 124.57 130.01 0 0 0 0 0 0 59.74 76.8 514384 185491 514384 185491 65.34 73.11 514384 202874 514384 176583 1720268 13.47 5.43 0 15.97 0 0.98 0 0.37 0 0.00 0 26.77 0 310496 0 43 0 41.76 0 1.40 0 0.00 0 1.14 0 0.00 0 111.06 0 0.33 0 23449 0 431919 0 68974 0 4225 0 1584 0 0 0 115614 0 10 0 0 0 85 0 8961 0 153 0 9209 0 55.92 0 241522 0 3659 9933 2.714676141022 431919.0 310496.0 23449.0 68974.0 4225.0 1584.0 0.0 115614.0 241522.0 71.9 5.4 16.0 1.0 0.4 0.0 26.8 55.9 43 43 43.00 38 18572517 26.7 21.6 21.4 30.2 0.0 35.5 23.8 smartseq 528686 SRR2088225 SRP060416 SRS980268 SRX1082194 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810660: T75_P2_B12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810660 GSM1810660 T75_P2_B12_ILC3 100952218 2347726 2016-01-28 01:00:06 113451947 100952218 2347726 1 2347726 index:0,count:2347726,average:43,stdev:0 GSM1810660_r1 2.43 8.86 0.25 57657850 68534924 44116985 55462725 118.86 125.72 0 0 0 0 0 0 52.52 70.3 2484178 748979 2484178 748979 57.81 67.6 2484178 824442 2484178 720300 10520036 18.25 7.55 0 15.36 0 1.12 0 0.28 0 0.00 0 37.85 0 1426136 0 43 0 41.41 0 1.31 0 0.00 0 1.29 0 0.01 0 291.44 0 0.35 0 177348 0 2347726 0 360658 0 26358 0 6585 0 0 0 888647 0 1 0 0 0 416 0 31449 0 878 0 32744 0 45.38 0 1065478 0 2900 36268 12.506206896552 2347726.0 1426136.0 177348.0 360658.0 26358.0 6585.0 0.0 888647.0 1065478.0 60.7 7.6 15.4 1.1 0.3 0.0 37.9 45.4 43 43 43.00 38 100952218 26.6 21.1 21.1 31.2 0.0 35.3 23.3 smartseq 528775 SRR2088230 SRP060416 SRS980263 SRX1082199 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810665: T75_P2_B6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810665 GSM1810665 T75_P2_B6_ILC3 95094199 2211493 2016-01-28 01:00:06 107844993 95094199 2211493 1 2211493 index:0,count:2211493,average:43,stdev:0 GSM1810665_r1 2.82 7.47 0.17 61443468 74981360 48250370 61908763 122.03 128.31 0 0 0 0 0 0 56.61 73.4 2581081 849102 2581081 849102 61.83 70.06 2581081 927340 2581081 810506 9455836 15.39 6.23 0 15.51 0 1.04 0 0.34 0 0.00 0 30.80 0 1499933 0 43 0 41.71 0 1.38 0 0.00 0 1.16 0 0.00 0 265.38 0 0.34 0 137810 0 2211493 0 343061 0 22976 0 7488 0 0 0 681096 0 10 0 0 0 326 0 34350 0 781 0 35467 0 52.31 0 1156872 0 4397 40303 9.166022287924 2211493.0 1499933.0 137810.0 343061.0 22976.0 7488.0 0.0 681096.0 1156872.0 67.8 6.2 15.5 1.0 0.3 0.0 30.8 52.3 43 43 43.00 38 95094199 26.4 21.5 21.7 30.4 0.0 35.1 23.4 smartseq 528823 SRR2088236 SRP060416 SRS980259 SRX1082205 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810671: T75_P2_C1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810671 GSM1810671 T75_P2_C1_ILC3 141233113 3284491 2016-01-28 01:00:06 157086029 141233113 3284491 1 3284491 index:0,count:3284491,average:43,stdev:0 GSM1810671_r1 3.71 6.21 0.22 95812506 119026411 75116526 98287289 124.23 130.85 0 0 0 0 0 0 58.71 75.91 4048372 1363126 4048372 1363126 64.76 72.61 4048372 1503602 4048372 1303845 13254327 13.83 5.63 0 16.02 0 1.02 0 0.38 0 0.00 0 27.91 0 2321908 0 43 0 41.83 0 1.45 0 0.00 0 1.11 0 0.00 0 369.51 0 0.32 0 185079 0 3284491 0 526271 0 33557 0 12391 0 0 0 916635 0 22 0 0 0 573 0 64913 0 1087 0 66595 0 54.67 0 1795637 0 5984 74684 12.480614973262 3284491.0 2321908.0 185079.0 526271.0 33557.0 12391.0 0.0 916635.0 1795637.0 70.7 5.6 16.0 1.0 0.4 0.0 27.9 54.7 43 43 43.00 38 141233113 26.4 22.0 22.0 29.7 0.0 35.6 24.0 smartseq 529031 SRR2088250 SRP060416 SRS980243 SRX1082219 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810685: T75_P2_D5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810685 GSM1810685 T75_P2_D5_ILC3 58122154 1351678 2016-01-28 01:00:06 65294620 58122154 1351678 1 1351678 index:0,count:1351678,average:43,stdev:0 GSM1810685_r1 2.77 6.86 0.19 36671091 44313261 28668669 36617079 120.84 127.73 0 0 0 0 0 0 58.14 75.61 1568060 519705 1568060 519705 63.03 71.78 1568060 563401 1568060 493377 5300799 14.45 6.61 0 15.28 0 0.97 0 0.28 0 0.00 0 32.62 0 893916 0 43 0 41.71 0 1.32 0 0.00 0 1.15 0 0.00 0 243.30 0 0.32 0 89315 0 1351678 0 206531 0 13067 0 3780 0 0 0 440915 0 19 0 0 0 203 0 24862 0 464 0 25548 0 50.85 0 687385 0 4872 27947 5.736247947455 1351678.0 893916.0 89315.0 206531.0 13067.0 3780.0 0.0 440915.0 687385.0 66.1 6.6 15.3 1.0 0.3 0.0 32.6 50.9 43 43 43.00 38 58122154 26.2 22.0 21.8 30.0 0.0 35.6 24.0 smartseq 529039 SRR2088251 SRP060416 SRS980244 SRX1082220 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810686: T75_P2_D6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810686 GSM1810686 T75_P2_D6_ILC3 67539412 1570684 2016-01-28 01:00:06 75596790 67539412 1570684 1 1570684 index:0,count:1570684,average:43,stdev:0 GSM1810686_r1 3.37 5.77 0.26 45737148 56537330 35820453 46875082 123.61 130.86 0 0 0 0 0 0 59.18 76.63 1969194 657012 1969194 657012 64.68 73.15 1969194 718006 1969194 627213 5954310 13.02 5.73 0 16.09 0 0.96 0 0.36 0 0.00 0 28.00 0 1110148 0 43 0 41.78 0 1.49 0 0.00 0 1.17 0 0.00 0 245.85 0 0.31 0 90071 0 1570684 0 252737 0 15086 0 5703 0 0 0 439747 0 28 0 0 0 242 0 31212 0 540 0 32022 0 54.59 0 857411 0 5917 35727 6.038026026703 1570684.0 1110148.0 90071.0 252737.0 15086.0 5703.0 0.0 439747.0 857411.0 70.7 5.7 16.1 1.0 0.4 0.0 28.0 54.6 43 43 43.00 38 67539412 26.3 22.1 21.9 29.7 0.0 35.7 24.1 smartseq 529295 SRR2088271 SRP060416 SRS980224 SRX1082240 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810706: T75_P2_F5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810706 GSM1810706 T75_P2_F5_ILC3 111961637 2603759 2016-01-28 01:00:06 125270991 111961637 2603759 1 2603759 index:0,count:2603759,average:43,stdev:0 GSM1810706_r1 2.8 6.34 0.19 72506523 87626105 57424408 72924369 120.85 126.99 0 0 0 0 0 0 58.25 74.73 3022028 1027474 3022028 1027474 62.66 70.92 3022028 1105382 3022028 975020 10699983 14.76 6.30 0 14.95 0 0.89 0 0.37 0 0.00 0 30.99 0 1764044 0 43 0 41.77 0 1.41 0 0.00 0 1.13 0 0.00 0 292.92 0 0.32 0 164087 0 2603759 0 389168 0 23129 0 9629 0 0 0 806957 0 26 0 0 0 396 0 42411 0 928 0 43761 0 52.80 0 1374876 0 4554 49255 10.815766359245 2603759.0 1764044.0 164087.0 389168.0 23129.0 9629.0 0.0 806957.0 1374876.0 67.7 6.3 14.9 0.9 0.4 0.0 31.0 52.8 43 43 43.00 38 111961637 26.4 21.9 21.8 29.9 0.0 35.5 23.9 smartseq 529303 SRR2088272 SRP060416 SRS980223 SRX1082241 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810707: T75_P2_F6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810707 GSM1810707 T75_P2_F6_ILC3 123952273 2882611 2016-01-28 01:00:06 138654833 123952273 2882611 1 2882611 index:0,count:2882611,average:43,stdev:0 GSM1810707_r1 2.82 6.18 0.24 86773248 107884287 71964488 93149230 124.33 129.44 0 0 0 0 0 0 59.85 73.16 3270775 1257465 3270775 1257465 63.28 69.63 3270775 1329370 3270775 1196726 13727121 15.82 5.29 0 13.26 0 0.91 0 0.46 0 0.00 0 25.75 0 2100901 0 43 0 41.87 0 1.40 0 0.00 0 1.14 0 0.00 0 334.75 0 0.32 0 152607 0 2882611 0 382158 0 26291 0 13222 0 0 0 742197 0 9 0 0 0 488 0 51565 0 1002 0 53064 0 59.62 0 1718743 0 5964 60042 10.067404426559 2882611.0 2100901.0 152607.0 382158.0 26291.0 13222.0 0.0 742197.0 1718743.0 72.9 5.3 13.3 0.9 0.5 0.0 25.7 59.6 43 43 43.00 38 123952273 26.4 22.0 22.0 29.6 0.0 35.5 24.0 smartseq 529311 SRR2088273 SRP060416 SRS980222 SRX1082242 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810708: T75_P2_F7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810708 GSM1810708 T75_P2_F7_ILC3 127710172 2970004 2016-01-28 01:00:06 142698497 127710172 2970004 1 2970004 index:0,count:2970004,average:43,stdev:0 GSM1810708_r1 3.61 7.12 0.18 84107049 104075739 67030106 86813933 123.74 129.51 0 0 0 0 0 0 62.25 79.39 3394203 1277780 3394203 1277780 66.7 75.2 3394203 1369076 3394203 1210265 10991955 13.07 5.97 0 14.92 0 1.00 0 0.43 0 0.00 0 29.46 0 2052654 0 43 0 41.65 0 1.37 0 0.00 0 1.14 0 0.00 0 237.60 0 0.33 0 177226 0 2970004 0 443216 0 29798 0 12691 0 0 0 874861 0 17 0 0 0 546 0 64625 0 1099 0 66287 0 54.19 0 1609438 0 5569 73021 13.112048841803 2970004.0 2052654.0 177226.0 443216.0 29798.0 12691.0 0.0 874861.0 1609438.0 69.1 6.0 14.9 1.0 0.4 0.0 29.5 54.2 43 43 43.00 38 127710172 26.4 21.9 21.8 30.0 0.0 35.5 23.8 smartseq 529319 SRR2088274 SRP060416 SRS980221 SRX1082243 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810709: T75_P2_F8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810709 GSM1810709 T75_P2_F8_ILC3 82366199 1915493 2016-01-28 01:00:06 92506963 82366199 1915493 1 1915493 index:0,count:1915493,average:43,stdev:0 GSM1810709_r1 4.16 4.88 0.24 66770397 84249922 55420618 72120154 126.18 130.13 0 0 0 0 0 0 62.07 75.43 2476282 995530 2476282 995530 66.87 71.91 2476282 1072549 2476282 949140 8643877 12.95 3.07 0 14.83 0 0.99 0 0.62 0 0.00 0 14.65 0 1603840 0 43 0 41.99 0 1.38 0 0.00 0 1.18 0 0.00 0 275.83 0 0.31 0 58814 0 1915493 0 284002 0 19037 0 11969 0 0 0 280647 0 9 0 0 0 446 0 48335 0 666 0 49456 0 68.90 0 1319838 0 5032 54918 10.913751987281 1915493.0 1603840.0 58814.0 284002.0 19037.0 11969.0 0.0 280647.0 1319838.0 83.7 3.1 14.8 1.0 0.6 0.0 14.7 68.9 43 43 43.00 38 82366199 27.3 21.7 21.9 29.1 0.0 35.6 24.4 smartseq 529335 SRR2088276 SRP060416 SRS980219 SRX1082245 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810711: T75_P2_G3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810711 GSM1810711 T75_P2_G3_ILC3 128706353 2993171 2016-01-28 01:00:06 142806793 128706353 2993171 1 2993171 index:0,count:2993171,average:43,stdev:0 GSM1810711_r1 4.84 5.53 0.19 93527512 118219972 76487961 100484774 126.4 131.37 0 0 0 0 0 0 59.75 73.89 3565426 1347380 3565426 1347380 64.41 70.34 3565426 1452485 3565426 1282637 13987039 14.95 4.68 0 14.42 0 1.02 0 0.55 0 0.00 0 23.09 0 2255174 0 43 0 41.94 0 1.33 0 0.00 0 1.17 0 0.00 0 239.45 0 0.31 0 140201 0 2993171 0 431579 0 30571 0 16388 0 0 0 691038 0 47 0 0 0 490 0 62236 0 1023 0 63796 0 60.93 0 1823595 0 7143 70098 9.813523729525 2993171.0 2255174.0 140201.0 431579.0 30571.0 16388.0 0.0 691038.0 1823595.0 75.3 4.7 14.4 1.0 0.5 0.0 23.1 60.9 43 43 43.00 38 128706353 26.5 22.2 22.1 29.3 0.0 35.8 24.4 smartseq 529343 SRR2088277 SRP060416 SRS979773 SRX1082246 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810712: T75_P2_G5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810712 GSM1810712 T75_P2_G5_ILC3 101056923 2350161 2016-01-28 01:00:06 112877284 101056923 2350161 1 2350161 index:0,count:2350161,average:43,stdev:0 GSM1810712_r1 3.44 6.7 0.26 67345674 81199725 54135235 68291358 120.57 126.15 0 0 0 0 0 0 55.26 69.86 2717998 905320 2717998 905320 59.54 66.56 2717998 975398 2717998 862465 12246373 18.18 5.88 0 14.57 0 1.05 0 0.44 0 0.00 0 28.80 0 1638180 0 43 0 41.78 0 1.34 0 0.00 0 1.16 0 0.01 0 52.88 0 0.32 0 138144 0 2350161 0 342323 0 24711 0 10458 0 0 0 676812 0 14 0 0 0 385 0 38814 0 842 0 40055 0 55.14 0 1295857 0 4997 44337 8.872723634181 2350161.0 1638180.0 138144.0 342323.0 24711.0 10458.0 0.0 676812.0 1295857.0 69.7 5.9 14.6 1.1 0.4 0.0 28.8 55.1 43 43 43.00 38 101056923 26.7 21.5 21.4 30.4 0.0 35.6 24.0 smartseq 529351 SRR2088278 SRP060416 SRS980218 SRX1082247 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810713: T75_P2_G6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810713 GSM1810713 T75_P2_G6_ILC3 127416482 2963174 2016-01-28 01:00:06 142731276 127416482 2963174 1 2963174 index:0,count:2963174,average:43,stdev:0 GSM1810713_r1 2.93 5.63 0.19 86584315 106189946 68276968 88506116 122.64 129.63 0 0 0 0 0 0 60.62 77.98 3671464 1274174 3671464 1274174 65.89 74.47 3671464 1385086 3671464 1216783 10715969 12.38 5.70 0 15.80 0 0.94 0 0.36 0 0.00 0 27.76 0 2102062 0 43 0 41.79 0 1.39 0 0.00 0 1.12 0 0.00 0 273.52 0 0.33 0 168999 0 2963174 0 468182 0 27986 0 10575 0 0 0 822551 0 24 0 0 0 546 0 61033 0 929 0 62532 0 55.14 0 1633880 0 6502 70282 10.809289449400 2963174.0 2102062.0 168999.0 468182.0 27986.0 10575.0 0.0 822551.0 1633880.0 70.9 5.7 15.8 0.9 0.4 0.0 27.8 55.1 43 43 43.00 38 127416482 26.3 22.0 22.0 29.7 0.0 35.5 23.8 smartseq 529359 SRR2088279 SRP060416 SRS980216 SRX1082248 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810714: T75_P2_G7_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810714 GSM1810714 T75_P2_G7_ILC3 102533414 2384498 2016-01-28 01:00:06 115394680 102533414 2384498 1 2384498 index:0,count:2384498,average:43,stdev:0 GSM1810714_r1 1.77 7.14 0.18 66052390 79744105 52950930 66992029 120.73 126.52 0 0 0 0 0 0 54.49 69.18 2663069 878319 2663069 878319 59.24 66.32 2663069 954958 2663069 842115 12001450 18.17 6.19 0 14.36 0 1.14 0 0.53 0 0.00 0 30.72 0 1612004 0 43 0 41.70 0 1.50 0 0.00 0 1.12 0 0.00 0 260.13 0 0.34 0 147509 0 2384498 0 342316 0 27295 0 12749 0 0 0 732450 0 6 0 0 0 336 0 34650 0 940 0 35932 0 53.25 0 1269688 0 3522 40212 11.417376490630 2384498.0 1612004.0 147509.0 342316.0 27295.0 12749.0 0.0 732450.0 1269688.0 67.6 6.2 14.4 1.1 0.5 0.0 30.7 53.2 43 43 43.00 38 102533414 26.7 21.5 21.5 30.3 0.0 35.4 23.6 smartseq 529438 SRR2088283 SRP060416 SRS980213 SRX1082252 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810718: T75_P2_H12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810718 GSM1810718 T75_P2_H12_ILC3 103827413 2414591 2016-01-28 01:00:06 116412751 103827413 2414591 1 2414591 index:0,count:2414591,average:43,stdev:0 GSM1810718_r1 2.78 7.5 0.21 67576501 83018831 53924976 69088738 122.85 128.12 0 0 0 0 0 0 59.27 75.56 2655336 978701 2655336 978701 63.62 71.5 2655336 1050519 2655336 926071 10071734 14.90 6.09 0 14.75 0 1.08 0 0.37 0 0.00 0 30.16 0 1651367 0 43 0 41.63 0 1.33 0 0.00 0 1.16 0 0.00 0 280.40 0 0.33 0 147128 0 2414591 0 356076 0 25989 0 9022 0 0 0 728213 0 1 0 0 0 284 0 44416 0 880 0 45581 0 53.64 0 1295291 0 4575 50826 11.109508196721 2414591.0 1651367.0 147128.0 356076.0 25989.0 9022.0 0.0 728213.0 1295291.0 68.4 6.1 14.7 1.1 0.4 0.0 30.2 53.6 43 43 43.00 38 103827413 26.6 21.5 21.3 30.5 0.0 35.4 23.7 smartseq 529454 SRR2088285 SRP060416 SRS980211 SRX1082254 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810720: T75_P2_H3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810720 GSM1810720 T75_P2_H3_ILC3 97734485 2272895 2016-01-28 01:00:06 109948742 97734485 2272895 1 2272895 index:0,count:2272895,average:43,stdev:0 GSM1810720_r1 2.84 7.25 0.31 62321613 76854600 49754147 64299356 123.32 129.23 0 0 0 0 0 0 58.26 74.26 2497165 885882 2497165 885882 62.19 70.18 2497165 945690 2497165 837212 9422785 15.12 6.48 0 14.41 0 0.93 0 0.35 0 0.00 0 31.82 0 1520616 0 43 0 41.70 0 1.33 0 0.00 0 1.12 0 0.00 0 215.33 0 0.33 0 147224 0 2272895 0 327598 0 21042 0 7940 0 0 0 723297 0 11 0 0 0 443 0 38518 0 739 0 39711 0 52.49 0 1193018 0 4642 44270 9.536837570013 2272895.0 1520616.0 147224.0 327598.0 21042.0 7940.0 0.0 723297.0 1193018.0 66.9 6.5 14.4 0.9 0.3 0.0 31.8 52.5 43 43 43.00 38 97734485 26.3 21.7 21.7 30.3 0.0 35.4 23.7 smartseq 529462 SRR2088286 SRP060416 SRS980210 SRX1082255 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810721: T75_P2_H4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810721 GSM1810721 T75_P2_H4_ILC3 115011756 2674692 2016-01-28 01:00:06 128820125 115011756 2674692 1 2674692 index:0,count:2674692,average:43,stdev:0 GSM1810721_r1 2.94 4.95 0.27 82442501 101647616 65728941 84828913 123.3 129.06 0 0 0 0 0 0 58.89 74.69 3401081 1168991 3401081 1168991 64.45 70.82 3401081 1279367 3401081 1108459 11042055 13.39 4.96 0 15.70 0 0.94 0 0.49 0 0.00 0 24.35 0 1985105 0 43 0 42.00 0 1.42 0 0.00 0 1.22 0 0.00 0 291.78 0 0.32 0 132629 0 2674692 0 419995 0 25158 0 13137 0 0 0 651292 0 24 0 0 0 388 0 56024 0 849 0 57285 0 58.52 0 1565110 0 6603 65642 9.941238830834 2674692.0 1985105.0 132629.0 419995.0 25158.0 13137.0 0.0 651292.0 1565110.0 74.2 5.0 15.7 0.9 0.5 0.0 24.4 58.5 43 43 43.00 38 115011756 26.3 22.3 22.3 29.1 0.0 35.5 24.2 smartseq 529471 SRR2088287 SRP060416 SRS980209 SRX1082256 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810722: T75_P2_H5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810722 GSM1810722 T75_P2_H5_ILC3 82301828 1913996 2016-01-28 01:00:06 92565172 82301828 1913996 1 1913996 index:0,count:1913996,average:43,stdev:0 GSM1810722_r1 2.89 4.76 0.23 62675293 78521993 49981366 65595912 125.28 131.24 0 0 0 0 0 0 60.07 76.07 2589063 907402 2589063 907402 65.9 72.09 2589063 995413 2589063 859916 7862594 12.54 4.15 0 16.59 0 1.03 0 0.43 0 0.00 0 19.63 0 1510454 0 43 0 41.90 0 1.45 0 0.00 0 1.16 0 0.00 0 362.65 0 0.32 0 79470 0 1913996 0 317614 0 19661 0 8208 0 0 0 375673 0 16 0 0 0 431 0 40085 0 642 0 41174 0 62.32 0 1192840 0 5531 46780 8.457783402640 1913996.0 1510454.0 79470.0 317614.0 19661.0 8208.0 0.0 375673.0 1192840.0 78.9 4.2 16.6 1.0 0.4 0.0 19.6 62.3 43 43 43.00 38 82301828 26.5 22.3 22.3 28.8 0.0 35.5 24.2 smartseq 529479 SRR2088288 SRP060416 SRS980208 SRX1082257 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810723: T75_P2_H9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810723 GSM1810723 T75_P2_H9_ILC3 22555951 524557 2016-01-28 01:00:06 25551358 22555951 524557 1 524557 index:0,count:524557,average:43,stdev:0 GSM1810723_r1 3.18 6.46 0.18 15266946 18370240 12296330 15451332 120.33 125.66 0 0 0 0 0 0 53.66 67.75 608752 199689 608752 199689 58.01 64.78 608752 215887 608752 190931 2783013 18.23 5.56 0 14.76 0 1.19 0 0.51 0 0.00 0 27.35 0 372172 0 43 0 41.72 0 1.47 0 0.01 0 1.15 0 0.00 0 111.08 0 0.33 0 29156 0 524557 0 77437 0 6253 0 2679 0 0 0 143453 0 0 0 0 0 103 0 7865 0 187 0 8155 0 56.19 0 294735 0 2514 9370 3.727128082737 524557.0 372172.0 29156.0 77437.0 6253.0 2679.0 0.0 143453.0 294735.0 70.9 5.6 14.8 1.2 0.5 0.0 27.3 56.2 43 43 43.00 38 22555951 27.3 21.1 21.0 30.6 0.0 35.5 23.7 smartseq 529551 SRR2088291 SRP060416 SRS980205 SRX1082260 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810726: T75_P3_A5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810726 GSM1810726 T75_P3_A5_ILC2 80516726 1872482 2016-01-28 01:00:06 91849029 80516726 1872482 1 1872482 index:0,count:1872482,average:43,stdev:0 GSM1810726_r1 5.04 8.82 0.27 50580497 62727428 40292526 52131828 124.02 129.38 0 0 0 0 0 0 57.96 74.41 1976010 720836 1976010 720836 62.77 71.66 1976010 780713 1976010 694213 8279100 16.37 7.30 0 14.69 0 1.02 0 0.34 0 0.00 0 32.22 0 1243772 0 43 0 41.59 0 1.25 0 0.01 0 1.12 0 0.00 0 187.25 0 0.36 0 136612 0 1872482 0 274978 0 19100 0 6365 0 0 0 603245 0 6 0 0 0 181 0 27343 0 685 0 28215 0 51.74 0 968794 0 4452 30794 6.916891284816 1872482.0 1243772.0 136612.0 274978.0 19100.0 6365.0 0.0 603245.0 968794.0 66.4 7.3 14.7 1.0 0.3 0.0 32.2 51.7 43 43 43.00 38 80516726 26.4 21.2 21.5 31.0 0.0 35.0 23.3 smartseq 529567 SRR2088293 SRP060416 SRS980203 SRX1082262 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810728: T75_P3_A8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810728 GSM1810728 T75_P3_A8_ILC2 52895547 1230129 2016-01-28 01:00:06 60590277 52895547 1230129 1 1230129 index:0,count:1230129,average:43,stdev:0 GSM1810728_r1 3.33 8.54 0.34 33503483 40851195 26346679 33476312 121.93 127.06 0 0 0 0 0 0 56.08 72.89 1344726 461872 1344726 461872 62.19 70.42 1344726 512150 1344726 446218 5549430 16.56 7.12 0 15.44 0 1.18 0 0.31 0 0.00 0 31.56 0 823562 0 43 0 41.58 0 1.29 0 0.01 0 1.20 0 0.00 0 201.29 0 0.36 0 87587 0 1230129 0 189941 0 14563 0 3764 0 0 0 388240 0 13 0 0 0 167 0 17974 0 480 0 18634 0 51.51 0 633621 0 3482 21638 6.214244686962 1230129.0 823562.0 87587.0 189941.0 14563.0 3764.0 0.0 388240.0 633621.0 66.9 7.1 15.4 1.2 0.3 0.0 31.6 51.5 43 43 43.00 38 52895547 26.8 21.0 21.3 31.0 0.0 35.0 23.3 smartseq 529574 SRR2088294 SRP060416 SRS980202 SRX1082263 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810729: T75_P3_B1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810729 GSM1810729 T75_P3_B1_ILC2 150667700 3503900 2016-01-28 01:00:06 167797369 150667700 3503900 1 3503900 index:0,count:3503900,average:43,stdev:0 GSM1810729_r1 4.36 9.18 0.27 90793110 112318746 72065017 93457812 123.71 129.69 0 0 0 0 0 0 55.66 71.75 3634913 1244760 3634913 1244760 60.43 69.27 3634913 1351393 3634913 1201634 16276552 17.93 7.86 0 14.31 0 1.19 0 0.37 0 0.00 0 34.62 0 2236366 0 43 0 41.54 0 1.32 0 0.01 0 1.14 0 0.00 0 323.44 0 0.34 0 275494 0 3503900 0 501559 0 41543 0 12955 0 0 0 1213036 0 5 0 0 0 334 0 42865 0 1544 0 44748 0 49.51 0 1734807 0 3614 47940 13.265080243498 3503900.0 2236366.0 275494.0 501559.0 41543.0 12955.0 0.0 1213036.0 1734807.0 63.8 7.9 14.3 1.2 0.4 0.0 34.6 49.5 43 43 43.00 38 150667700 26.2 21.4 21.4 30.9 0.0 35.4 23.8 smartseq 529591 SRR2088296 SRP060416 SRS980199 SRX1082265 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810731: T75_P3_B4_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810731 GSM1810731 T75_P3_B4_NK 123696724 2876668 2016-01-28 01:00:06 138142429 123696724 2876668 1 2876668 index:0,count:2876668,average:43,stdev:0 GSM1810731_r1 3.81 7.21 0.3 82076891 102261617 65142469 83962983 124.59 128.89 0 0 0 0 0 0 57.01 73.02 3225589 1139127 3225589 1139127 63.28 69.58 3225589 1264405 3225589 1085374 13825503 16.84 6.49 0 15.24 0 1.17 0 0.46 0 0.00 0 28.90 0 1998262 0 43 0 41.76 0 1.19 0 0.01 0 1.16 0 0.00 0 240.84 0 0.33 0 186667 0 2876668 0 438265 0 33547 0 13369 0 0 0 831490 0 2 0 0 0 325 0 49298 0 1091 0 50716 0 54.23 0 1559997 0 5187 58829 11.341623288992 2876668.0 1998262.0 186667.0 438265.0 33547.0 13369.0 0.0 831490.0 1559997.0 69.5 6.5 15.2 1.2 0.5 0.0 28.9 54.2 43 43 43.00 38 123696724 26.3 21.8 22.0 29.9 0.0 35.5 24.3 smartseq 529598 SRR2088297 SRP060416 SRS980201 SRX1082266 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810732: T75_P3_B5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810732 GSM1810732 T75_P3_B5_ILC2 150970291 3510937 2016-01-28 01:00:06 168118340 150970291 3510937 1 3510937 index:0,count:3510937,average:43,stdev:0 GSM1810732_r1 4.39 7.14 0.25 103500389 131420776 82652790 108814380 126.98 131.65 0 0 0 0 0 0 56.78 72.26 3986544 1429189 3986544 1429189 62.63 68.94 3986544 1576399 3986544 1363489 17319612 16.73 6.03 0 15.36 0 1.10 0 0.43 0 0.00 0 26.79 0 2517022 0 43 0 41.79 0 1.24 0 0.01 0 1.14 0 0.00 0 324.09 0 0.33 0 211764 0 3510937 0 539147 0 38546 0 14942 0 0 0 940427 0 44 0 0 0 243 0 56845 0 1324 0 58456 0 56.33 0 1977875 0 4945 67271 13.603842264914 3510937.0 2517022.0 211764.0 539147.0 38546.0 14942.0 0.0 940427.0 1977875.0 71.7 6.0 15.4 1.1 0.4 0.0 26.8 56.3 43 43 43.00 38 150970291 26.4 21.8 21.9 29.9 0.0 35.5 24.3 smartseq 529606 SRR2088298 SRP060416 SRS980198 SRX1082267 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810733: T75_P3_B6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810733 GSM1810733 T75_P3_B6_ILC2 124112964 2886348 2016-01-28 01:00:06 140396367 124112964 2886348 1 2886348 index:0,count:2886348,average:43,stdev:0 GSM1810733_r1 5.38 8.31 0.24 78461409 97797194 61679386 80245359 124.64 130.1 0 0 0 0 0 0 54.03 70.11 3112710 1036727 3112710 1036727 60.04 67.55 3112710 1152013 3112710 998930 14496732 18.48 7.24 0 15.25 0 1.12 0 0.49 0 0.00 0 31.91 0 1918814 0 43 0 41.71 0 1.21 0 0.01 0 1.14 0 0.00 0 259.77 0 0.36 0 208940 0 2886348 0 440059 0 32450 0 14025 0 0 0 921059 0 13 0 0 0 198 0 38673 0 1145 0 40029 0 51.23 0 1478755 0 4945 46603 9.424266936299 2886348.0 1918814.0 208940.0 440059.0 32450.0 14025.0 0.0 921059.0 1478755.0 66.5 7.2 15.2 1.1 0.5 0.0 31.9 51.2 43 43 43.00 38 124112964 26.5 21.2 21.5 30.7 0.0 35.1 23.6 smartseq 529615 SRR2088299 SRP060416 SRS980197 SRX1082268 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810734: T75_P3_B7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810734 GSM1810734 T75_P3_B7_ILC2 135991069 3162583 2016-01-28 01:00:06 151774177 135991069 3162583 1 3162583 index:0,count:3162583,average:43,stdev:0 GSM1810734_r1 4.72 7.36 0.22 91338223 115211988 72858149 95137718 126.14 130.58 0 0 0 0 0 0 57.49 73.29 3516249 1280357 3516249 1280357 63.48 69.88 3516249 1413902 3516249 1220824 14105367 15.44 6.40 0 15.18 0 1.06 0 0.41 0 0.00 0 28.11 0 2227178 0 43 0 41.71 0 1.24 0 0.01 0 1.16 0 0.00 0 355.79 0 0.33 0 202340 0 3162583 0 480238 0 33501 0 12836 0 0 0 889068 0 51 0 0 0 311 0 53647 0 1209 0 55218 0 55.24 0 1746940 0 5109 61745 12.085535329810 3162583.0 2227178.0 202340.0 480238.0 33501.0 12836.0 0.0 889068.0 1746940.0 70.4 6.4 15.2 1.1 0.4 0.0 28.1 55.2 43 43 43.00 38 135991069 26.4 21.7 21.6 30.2 0.0 35.5 24.1 smartseq 530438 SRR2088300 SRP060416 SRS980196 SRX1082269 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810735: T75_P3_B8_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810735 GSM1810735 T75_P3_B8_ILC2 26830839 623973 2016-01-28 01:00:06 31329858 26830839 623973 1 623973 index:0,count:623973,average:43,stdev:0 GSM1810735_r1 3.64 8.73 0.3 17515600 21483792 13769751 17719364 122.66 128.68 0 0 0 0 0 0 56.39 73.38 706195 243836 706195 243836 61.8 70.74 706195 267219 706195 235049 3030429 17.30 6.61 0 16.05 0 1.18 0 0.34 0 0.00 0 29.18 0 432390 0 43 0 41.44 0 1.27 0 0.01 0 1.13 0 0.00 0 124.79 0 0.41 0 41272 0 623973 0 100117 0 7361 0 2119 0 0 0 182103 0 4 0 0 0 66 0 8706 0 216 0 8992 0 53.25 0 332273 0 2714 9608 3.540162122329 623973.0 432390.0 41272.0 100117.0 7361.0 2119.0 0.0 182103.0 332273.0 69.3 6.6 16.0 1.2 0.3 0.0 29.2 53.3 43 43 43.00 38 26830839 27.0 20.4 21.2 31.5 0.0 34.4 22.5 smartseq 530446 SRR2088301 SRP060416 SRS980195 SRX1082270 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810736: T75_P3_C10_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810736 GSM1810736 T75_P3_C10_ILC2 37485035 871745 2016-01-28 01:00:06 42984579 37485035 871745 1 871745 index:0,count:871745,average:43,stdev:0 GSM1810736_r1 8.11 6.67 0.33 27813360 36981775 22540169 30905888 132.96 137.11 0 0 0 0 0 0 61.91 77.43 1022058 417104 1022058 417104 68.14 73.88 1022058 459099 1022058 397988 3626745 13.04 4.89 0 15.49 0 0.90 0 0.35 0 0.00 0 21.47 0 673751 0 43 0 41.84 0 1.17 0 0.01 0 1.12 0 0.00 0 209.22 0 0.35 0 42648 0 871745 0 135076 0 7818 0 3052 0 0 0 187124 0 5 0 0 0 111 0 17885 0 271 0 18272 0 61.79 0 538675 0 4954 20514 4.140896245458 871745.0 673751.0 42648.0 135076.0 7818.0 3052.0 0.0 187124.0 538675.0 77.3 4.9 15.5 0.9 0.4 0.0 21.5 61.8 43 43 43.00 38 37485035 27.0 21.4 21.7 30.0 0.0 35.2 23.9 smartseq 530455 SRR2088302 SRP060416 SRS979774 SRX1082271 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810737: T75_P3_C11_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810737 GSM1810737 T75_P3_C11_ILC2 138344674 3217318 2016-01-28 01:00:06 154187324 138344674 3217318 1 3217318 index:0,count:3217318,average:43,stdev:0 GSM1810737_r1 5.09 7.82 0.32 92991014 118471797 72368846 95902215 127.4 132.52 0 0 0 0 0 0 58.97 77.06 3732546 1339453 3732546 1339453 65.76 73.37 3732546 1493691 3732546 1275238 13059424 14.04 6.29 0 16.58 0 1.13 0 0.38 0 0.00 0 27.89 0 2271488 0 43 0 41.64 0 1.17 0 0.01 0 1.12 0 0.00 0 263.24 0 0.33 0 202516 0 3217318 0 533331 0 36398 0 12201 0 0 0 897231 0 6 0 0 0 403 0 60654 0 1186 0 62249 0 54.03 0 1738157 0 5605 71384 12.735771632471 3217318.0 2271488.0 202516.0 533331.0 36398.0 12201.0 0.0 897231.0 1738157.0 70.6 6.3 16.6 1.1 0.4 0.0 27.9 54.0 43 43 43.00 38 138344674 26.5 21.6 21.6 30.4 0.0 35.5 24.1 smartseq 530462 SRR2088303 SRP060416 SRS980194 SRX1082272 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810738: T75_P3_C12_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810738 GSM1810738 T75_P3_C12_ILC2 107702831 2504717 2016-01-28 01:00:06 121179727 107702831 2504717 1 2504717 index:0,count:2504717,average:43,stdev:0 GSM1810738_r1 4.2 7.99 0.26 69793109 85944338 55516599 71538658 123.14 128.86 0 0 0 0 0 0 57.28 73.5 2775189 982080 2775189 982080 62.62 71.13 2775189 1073653 2775189 950390 11753214 16.84 6.88 0 15.10 0 1.16 0 0.35 0 0.00 0 30.04 0 1714444 0 43 0 41.55 0 1.36 0 0.01 0 1.13 0 0.00 0 300.57 0 0.35 0 172383 0 2504717 0 378261 0 28971 0 8851 0 0 0 752451 0 31 0 0 0 327 0 38807 0 986 0 40151 0 53.35 0 1336183 0 3942 44270 11.230339928970 2504717.0 1714444.0 172383.0 378261.0 28971.0 8851.0 0.0 752451.0 1336183.0 68.4 6.9 15.1 1.2 0.4 0.0 30.0 53.3 43 43 43.00 38 107702831 26.8 21.1 21.1 30.9 0.0 35.3 23.7 smartseq 530471 SRR2088304 SRP060416 SRS980193 SRX1082273 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810739: T75_P3_C2_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810739 GSM1810739 T75_P3_C2_ILC2 36301460 844220 2016-01-28 01:00:06 41816910 36301460 844220 1 844220 index:0,count:844220,average:43,stdev:0 GSM1810739_r1 6.51 6.9 0.26 25807752 34307634 20648212 28574979 132.94 138.39 0 0 0 0 0 0 60.19 76.4 990186 377872 990186 377872 66.47 72.69 990186 417339 990186 359536 3424097 13.27 5.55 0 15.78 0 1.03 0 0.38 0 0.00 0 24.22 0 627825 0 43 0 41.75 0 1.21 0 0.01 0 1.14 0 0.00 0 159.96 0 0.37 0 46826 0 844220 0 133218 0 8723 0 3224 0 0 0 204448 0 14 0 0 0 93 0 15263 0 316 0 15686 0 58.59 0 494607 0 4799 17619 3.671389872890 844220.0 627825.0 46826.0 133218.0 8723.0 3224.0 0.0 204448.0 494607.0 74.4 5.5 15.8 1.0 0.4 0.0 24.2 58.6 43 43 43.00 38 36301460 26.6 21.4 22.0 30.0 0.0 35.0 23.6 smartseq 530487 SRR2088306 SRP060416 SRS980191 SRX1082275 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810741: T75_P3_C4_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810741 GSM1810741 T75_P3_C4_ILC2 113787847 2646229 2016-01-28 01:00:06 127326347 113787847 2646229 1 2646229 index:0,count:2646229,average:43,stdev:0 GSM1810741_r1 4.19 9.54 0.25 66149123 80550137 52197211 66503368 121.77 127.41 0 0 0 0 0 0 54.94 71.29 2650218 895721 2650218 895721 60.04 69.03 2650218 979006 2650218 867282 12012587 18.16 8.40 0 14.13 0 1.07 0 0.32 0 0.00 0 37.00 0 1630486 0 43 0 41.54 0 1.20 0 0.01 0 1.15 0 0.00 0 238.16 0 0.33 0 222158 0 2646229 0 374027 0 28256 0 8479 0 0 0 979008 0 0 0 0 0 221 0 32071 0 925 0 33217 0 47.48 0 1256459 0 3652 37023 10.137732749179 2646229.0 1630486.0 222158.0 374027.0 28256.0 8479.0 0.0 979008.0 1256459.0 61.6 8.4 14.1 1.1 0.3 0.0 37.0 47.5 43 43 43.00 38 113787847 26.3 21.4 21.5 30.8 0.0 35.4 23.9 smartseq 530959 SRR2088341 SRP060416 SRS980157 SRX1082310 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810776: T75_P3_G1_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810776 GSM1810776 T75_P3_G1_ILC2 131416127 3056189 2016-01-28 01:00:06 145958539 131416127 3056189 1 3056189 index:0,count:3056189,average:43,stdev:0 GSM1810776_r1 4.83 7.71 0.41 88804331 111683933 73507207 95591721 125.76 130.04 0 0 0 0 0 0 59.55 73.18 3235686 1288678 3235686 1288678 63.92 70.61 3235686 1383293 3235686 1243398 13238262 14.91 6.24 0 13.19 0 1.12 0 0.52 0 0.00 0 27.55 0 2163968 0 43 0 41.74 0 1.22 0 0.01 0 1.15 0 0.00 0 255.87 0 0.32 0 190793 0 3056189 0 402986 0 34224 0 15888 0 0 0 842109 0 30 0 0 0 352 0 46113 0 1209 0 47704 0 57.62 0 1760982 0 5000 54166 10.833200000000 3056189.0 2163968.0 190793.0 402986.0 34224.0 15888.0 0.0 842109.0 1760982.0 70.8 6.2 13.2 1.1 0.5 0.0 27.6 57.6 43 43 43.00 38 131416127 26.5 21.6 21.5 30.3 0.0 35.7 24.3 smartseq 530967 SRR2088342 SRP060416 SRS980156 SRX1082311 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810777: T75_P3_G3_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810777 GSM1810777 T75_P3_G3_ILC2 127781853 2971671 2016-01-28 01:00:06 141669051 127781853 2971671 1 2971671 index:0,count:2971671,average:43,stdev:0 GSM1810777_r1 4.94 7.68 0.32 82895613 104016322 67104884 87106373 125.48 129.81 0 0 0 0 0 0 58.75 73.89 3120484 1188759 3120484 1188759 63.83 70.94 3120484 1291533 3120484 1141386 12710488 15.33 6.89 0 13.95 0 1.07 0 0.38 0 0.00 0 30.45 0 2023455 0 43 0 41.71 0 1.28 0 0.01 0 1.11 0 0.00 0 334.31 0 0.32 0 204645 0 2971671 0 414554 0 31885 0 11373 0 0 0 904958 0 2 0 0 0 321 0 44011 0 1096 0 45430 0 54.14 0 1608901 0 5259 50589 9.619509412436 2971671.0 2023455.0 204645.0 414554.0 31885.0 11373.0 0.0 904958.0 1608901.0 68.1 6.9 14.0 1.1 0.4 0.0 30.5 54.1 43 43 43.00 38 127781853 26.4 21.7 21.6 30.3 0.0 35.7 24.4 smartseq 530975 SRR2088343 SRP060416 SRS980155 SRX1082312 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810778: T75_P3_G5_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810778 GSM1810778 T75_P3_G5_ILC2 135245320 3145240 2016-01-28 01:00:06 150401007 135245320 3145240 1 3145240 index:0,count:3145240,average:43,stdev:0 GSM1810778_r1 4.69 7.66 0.23 84597262 102012836 67862814 84816973 120.59 124.98 0 0 0 0 0 0 56.25 71.5 3285722 1162779 3285722 1162779 61.13 68.71 3285722 1263657 3285722 1117425 14787521 17.48 7.42 0 14.01 0 1.07 0 0.41 0 0.00 0 32.80 0 2067058 0 43 0 41.73 0 1.14 0 0.01 0 1.15 0 0.01 0 251.62 0 0.32 0 233432 0 3145240 0 440724 0 33606 0 12861 0 0 0 1031715 0 21 0 0 0 314 0 41666 0 1133 0 43134 0 51.71 0 1626334 0 4560 51075 11.200657894737 3145240.0 2067058.0 233432.0 440724.0 33606.0 12861.0 0.0 1031715.0 1626334.0 65.7 7.4 14.0 1.1 0.4 0.0 32.8 51.7 43 43 43.00 38 135245320 26.3 21.6 21.7 30.4 0.0 35.6 24.2 smartseq 530983 SRR2088344 SRP060416 SRS980154 SRX1082313 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810779: T75_P3_G6_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810779 GSM1810779 T75_P3_G6_ILC2 145023692 3372644 2016-01-28 01:00:06 161777091 145023692 3372644 1 3372644 index:0,count:3372644,average:43,stdev:0 GSM1810779_r1 3.42 6.41 0.23 105923569 135335910 86800996 113890841 127.77 131.21 0 0 0 0 0 0 61.84 76.43 3857542 1583990 3857542 1583990 67.46 72.88 3857542 1728030 3857542 1510299 14528698 13.72 5.18 0 14.50 0 0.96 0 0.34 0 0.00 0 22.75 0 2561524 0 43 0 41.88 0 1.21 0 0.01 0 1.14 0 0.00 0 263.95 0 0.31 0 174651 0 3372644 0 489123 0 32439 0 11521 0 0 0 767160 0 20 0 0 0 426 0 74244 0 1289 0 75979 0 61.45 0 2072401 0 7647 85703 11.207401595397 3372644.0 2561524.0 174651.0 489123.0 32439.0 11521.0 0.0 767160.0 2072401.0 76.0 5.2 14.5 1.0 0.3 0.0 22.7 61.4 43 43 43.00 38 145023692 26.5 21.9 22.0 29.6 0.0 35.6 24.5 smartseq 530991 SRR2088345 SRP060416 SRS980153 SRX1082314 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810780: T75_P3_G7_ILC2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC2|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810780 GSM1810780 T75_P3_G7_ILC2 95443144 2219608 2016-01-28 01:00:06 107425713 95443144 2219608 1 2219608 index:0,count:2219608,average:43,stdev:0 GSM1810780_r1 5.96 8.27 0.31 60285999 75415203 47742776 62066196 125.1 130.0 0 0 0 0 0 0 58.08 74.84 2348697 859332 2348697 859332 63.65 71.72 2348697 941784 2348697 823616 9059526 15.03 7.22 0 14.92 0 1.12 0 0.32 0 0.00 0 31.90 0 1479519 0 43 0 41.58 0 1.16 0 0.01 0 1.15 0 0.01 0 266.35 0 0.34 0 160325 0 2219608 0 331221 0 24903 0 7066 0 0 0 708120 0 3 0 0 0 308 0 32489 0 823 0 33623 0 51.73 0 1148298 0 4201 36981 8.802904070459 2219608.0 1479519.0 160325.0 331221.0 24903.0 7066.0 0.0 708120.0 1148298.0 66.7 7.2 14.9 1.1 0.3 0.0 31.9 51.7 43 43 43.00 38 95443144 26.6 21.3 21.2 30.9 0.0 35.4 23.8 smartseq 531383 SRR2088376 SRP060416 SRS980123 SRX1082345 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810811: T75_P4_C6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810811 GSM1810811 T75_P4_C6_ILC1 146796152 3413864 2016-01-28 01:00:06 160815560 146796152 3413864 1 3413864 index:0,count:3413864,average:43,stdev:0 GSM1810811_r1 1.73 4.44 0.17 107527944 128826272 79697630 103809839 119.81 130.25 0 0 0 0 0 0 55.01 75.09 5443722 1427958 5443722 1427958 61.98 71.21 5443722 1608694 5443722 1354100 13738777 12.78 4.62 0 20.33 0 1.12 0 0.39 0 0.00 0 22.46 0 2595693 0 43 0 41.91 0 1.39 0 0.00 0 1.17 0 0.00 0 332.16 0 0.28 0 157757 0 3413864 0 694001 0 38211 0 13285 0 0 0 766675 0 7 0 0 0 534 0 61951 0 1088 0 63580 0 55.70 0 1901692 0 7482 73797 9.863271852446 3413864.0 2595693.0 157757.0 694001.0 38211.0 13285.0 0.0 766675.0 1901692.0 76.0 4.6 20.3 1.1 0.4 0.0 22.5 55.7 43 43 43.00 38 146796152 26.2 22.2 22.3 29.3 0.0 36.1 25.2 smartseq 531391 SRR2088377 SRP060416 SRS980121 SRX1082346 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810812: T75_P4_C7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810812 GSM1810812 T75_P4_C7_ILC1 132392829 3078903 2016-01-28 01:00:06 144907039 132392829 3078903 1 3078903 index:0,count:3078903,average:43,stdev:0 GSM1810812_r1 1.8 4.67 0.21 98541870 120044089 74208278 97663336 121.82 131.61 0 0 0 0 0 0 57.41 77.09 4794713 1367379 4794713 1367379 64.14 72.86 4794713 1527805 4794713 1292402 12627704 12.81 4.33 0 19.75 0 1.18 0 0.46 0 0.00 0 21.01 0 2381919 0 43 0 41.84 0 1.41 0 0.00 0 1.11 0 0.00 0 326.00 0 0.28 0 133218 0 3078903 0 608153 0 36184 0 14075 0 0 0 646725 0 15 0 0 0 647 0 65568 0 1024 0 67254 0 57.61 0 1773766 0 7927 78073 9.848997098524 3078903.0 2381919.0 133218.0 608153.0 36184.0 14075.0 0.0 646725.0 1773766.0 77.4 4.3 19.8 1.2 0.5 0.0 21.0 57.6 43 43 43.00 38 132392829 26.2 22.3 22.3 29.1 0.0 36.1 25.2 smartseq 531399 SRR2088378 SRP060416 SRS980122 SRX1082347 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810813: T75_P4_C8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810813 GSM1810813 T75_P4_C8_ILC1 98301741 2286087 2016-01-28 01:00:06 107975578 98301741 2286087 1 2286087 index:0,count:2286087,average:43,stdev:0 GSM1810813_r1 2.13 4.17 0.23 85813444 111717253 67348768 91886344 130.19 136.43 0 0 0 0 0 0 65.79 84.29 3638348 1352043 3638348 1352043 73.09 78.95 3638348 1502043 3638348 1266442 6656637 7.76 1.85 0 19.73 0 1.06 0 0.40 0 0.00 0 8.64 0 2055191 0 43 0 41.99 0 1.39 0 0.00 0 1.16 0 0.00 0 316.54 0 0.27 0 42311 0 2286087 0 451157 0 24210 0 9119 0 0 0 197567 0 34 0 0 0 641 0 71277 0 686 0 72638 0 70.17 0 1604034 0 9765 86724 8.881105990783 2286087.0 2055191.0 42311.0 451157.0 24210.0 9119.0 0.0 197567.0 1604034.0 89.9 1.9 19.7 1.1 0.4 0.0 8.6 70.2 43 43 43.00 38 98301741 26.5 22.6 22.8 28.1 0.0 36.3 25.9 smartseq 531406 SRR2088379 SRP060416 SRS980120 SRX1082348 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810814: T75_P4_C9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810814 GSM1810814 T75_P4_C9_ILC1 34076769 792483 2016-01-28 01:00:06 37721149 34076769 792483 1 792483 index:0,count:792483,average:43,stdev:0 GSM1810814_r1 2.41 5.12 0.27 25990497 32122135 19949504 26379229 123.59 132.23 0 0 0 0 0 0 57.19 75.29 1195955 358413 1195955 358413 64.03 71.97 1195955 401259 1195955 342598 3340960 12.85 3.93 0 19.01 0 1.14 0 0.72 0 0.00 0 19.06 0 626700 0 43 0 41.91 0 1.42 0 0.01 0 1.11 0 0.00 0 203.78 0 0.29 0 31124 0 792483 0 150671 0 9023 0 5684 0 0 0 151076 0 10 0 0 0 190 0 15825 0 263 0 16288 0 60.07 0 476029 0 5484 18971 3.459336250912 792483.0 626700.0 31124.0 150671.0 9023.0 5684.0 0.0 151076.0 476029.0 79.1 3.9 19.0 1.1 0.7 0.0 19.1 60.1 43 43 43.00 38 34076769 26.6 22.1 22.1 29.3 0.0 36.2 25.4 smartseq 531462 SRR2088380 SRP060416 SRS980119 SRX1082349 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810815: T75_P4_D10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810815 GSM1810815 T75_P4_D10_ILC1 41417256 963192 2016-01-28 01:00:06 45961979 41417256 963192 1 963192 index:0,count:963192,average:43,stdev:0 GSM1810815_r1 1.09 7.18 0.23 24999880 29577061 18137939 23045612 118.31 127.06 0 0 0 0 0 0 54.19 76.15 1223976 332915 1223976 332915 61.42 72.26 1223976 377326 1223976 315876 3735136 14.94 6.93 0 18.40 0 1.15 0 0.33 0 0.00 0 34.74 0 614346 0 43 0 41.49 0 1.44 0 0.00 0 1.14 0 0.00 0 266.73 0 0.31 0 66771 0 963192 0 177190 0 11041 0 3153 0 0 0 334652 0 7 0 0 0 135 0 15005 0 366 0 15513 0 45.39 0 437156 0 4295 18557 4.320605355064 963192.0 614346.0 66771.0 177190.0 11041.0 3153.0 0.0 334652.0 437156.0 63.8 6.9 18.4 1.1 0.3 0.0 34.7 45.4 43 43 43.00 38 41417256 25.9 21.8 21.7 30.5 0.0 36.0 24.6 smartseq 531471 SRR2088381 SRP060416 SRS980118 SRX1082350 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810816: T75_P4_D11_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810816 GSM1810816 T75_P4_D11_NK 98238273 2284611 2016-01-28 01:00:06 107830203 98238273 2284611 1 2284611 index:0,count:2284611,average:43,stdev:0 GSM1810816_r1 2.4 5.07 0.24 73906373 90573891 58825869 75813367 122.55 128.88 0 0 0 0 0 0 56.77 72.07 3121783 1011471 3121783 1011471 62.18 68.3 3121783 1108020 3121783 958485 10327912 13.97 4.18 0 16.56 0 1.11 0 0.53 0 0.00 0 20.37 0 1781827 0 43 0 41.92 0 1.34 0 0.00 0 1.18 0 0.00 0 432.87 0 0.28 0 95486 0 2284611 0 378423 0 25386 0 12122 0 0 0 465276 0 9 0 0 0 538 0 45729 0 774 0 47050 0 61.43 0 1403404 0 6191 54106 8.739460507188 2284611.0 1781827.0 95486.0 378423.0 25386.0 12122.0 0.0 465276.0 1403404.0 78.0 4.2 16.6 1.1 0.5 0.0 20.4 61.4 43 43 43.00 38 98238273 26.5 22.2 22.2 29.0 0.0 36.2 25.6 smartseq 531477 SRR2088382 SRP060416 SRS980117 SRX1082351 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810817: T75_P4_D12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810817 GSM1810817 T75_P4_D12_ILC1 97257787 2261809 2016-01-28 01:00:06 106915353 97257787 2261809 1 2261809 index:0,count:2261809,average:43,stdev:0 GSM1810817_r1 2.12 4.93 0.24 73655710 92561123 57901733 76658602 125.67 132.39 0 0 0 0 0 0 60.75 78.07 3167923 1079616 3167923 1079616 66.53 73.36 3167923 1182447 3167923 1014473 8715475 11.83 4.12 0 17.43 0 1.02 0 0.42 0 0.00 0 19.98 0 1777190 0 43 0 41.87 0 1.37 0 0.00 0 1.17 0 0.00 0 271.42 0 0.28 0 93179 0 2261809 0 394278 0 23065 0 9591 0 0 0 451963 0 31 0 0 0 458 0 52214 0 817 0 53520 0 61.14 0 1382912 0 7655 61887 8.084519921620 2261809.0 1777190.0 93179.0 394278.0 23065.0 9591.0 0.0 451963.0 1382912.0 78.6 4.1 17.4 1.0 0.4 0.0 20.0 61.1 43 43 43.00 38 97257787 26.3 22.4 22.4 28.9 0.0 36.2 25.5 smartseq 531485 SRR2088383 SRP060416 SRS980115 SRX1082352 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810818: T75_P4_D2_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810818 GSM1810818 T75_P4_D2_NK 45191882 1050974 2016-01-28 01:00:06 50029824 45191882 1050974 1 1050974 index:0,count:1050974,average:43,stdev:0 GSM1810818_r1 2.01 6.19 0.17 28740642 33993301 21570846 27521853 118.28 127.59 0 0 0 0 0 0 52.97 71.79 1394991 371784 1394991 371784 58.46 68.25 1394991 410277 1394991 353500 4473907 15.57 6.38 0 17.50 0 1.21 0 0.43 0 0.00 0 31.58 0 701814 0 43 0 41.65 0 1.43 0 0.00 0 1.16 0 0.00 0 222.56 0 0.30 0 67025 0 1050974 0 183902 0 12744 0 4472 0 0 0 331944 0 7 0 0 0 144 0 16185 0 400 0 16736 0 49.28 0 517912 0 4319 19229 4.452188006483 1050974.0 701814.0 67025.0 183902.0 12744.0 4472.0 0.0 331944.0 517912.0 66.8 6.4 17.5 1.2 0.4 0.0 31.6 49.3 43 43 43.00 38 45191882 26.0 22.0 21.9 30.1 0.0 36.1 24.9 smartseq 531493 SRR2088384 SRP060416 SRS980114 SRX1082353 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810819: T75_P4_D3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810819 GSM1810819 T75_P4_D3_ILC1 80692768 1876576 2016-01-28 01:00:06 88746852 80692768 1876576 1 1876576 index:0,count:1876576,average:43,stdev:0 GSM1810819_r1 2.31 4.05 0.15 61234543 75448466 43962789 59424681 123.21 135.17 0 0 0 0 0 0 55.9 78.54 3228741 823813 3228741 823813 64.71 74.18 3228741 953686 3228741 778098 7307961 11.93 4.15 0 22.64 0 1.11 0 0.37 0 0.00 0 19.98 0 1473697 0 43 0 41.91 0 1.43 0 0.00 0 1.14 0 0.00 0 241.27 0 0.29 0 77871 0 1876576 0 424789 0 20912 0 6976 0 0 0 374991 0 14 0 0 0 342 0 40935 0 542 0 41833 0 55.89 0 1048908 0 7532 49837 6.616702071163 1876576.0 1473697.0 77871.0 424789.0 20912.0 6976.0 0.0 374991.0 1048908.0 78.5 4.1 22.6 1.1 0.4 0.0 20.0 55.9 43 43 43.00 38 80692768 26.1 22.6 22.6 28.7 0.0 36.3 25.7 smartseq 531501 SRR2088385 SRP060416 SRS980116 SRX1082354 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810820: T75_P4_D5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810820 GSM1810820 T75_P4_D5_ILC1 71503066 1662862 2016-01-28 01:00:06 78915425 71503066 1662862 1 1662862 index:0,count:1662862,average:43,stdev:0 GSM1810820_r1 2.3 4.53 0.21 50793253 60677937 37978340 49284308 119.46 129.77 0 0 0 0 0 0 55.26 74.84 2515508 679511 2515508 679511 61.8 71.06 2515508 759946 2515508 645209 6941876 13.67 5.02 0 19.34 0 1.20 0 0.45 0 0.00 0 24.40 0 1229658 0 43 0 41.83 0 1.47 0 0.00 0 1.11 0 0.00 0 65.78 0 0.29 0 83429 0 1662862 0 321663 0 19893 0 7547 0 0 0 405764 0 18 0 0 0 333 0 27172 0 582 0 28105 0 54.60 0 907995 0 5918 33908 5.729638391348 1662862.0 1229658.0 83429.0 321663.0 19893.0 7547.0 0.0 405764.0 907995.0 73.9 5.0 19.3 1.2 0.5 0.0 24.4 54.6 43 43 43.00 38 71503066 26.3 22.1 22.1 29.4 0.0 36.1 25.2 smartseq 531509 SRR2088386 SRP060416 SRS980113 SRX1082355 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810821: T75_P4_D6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810821 GSM1810821 T75_P4_D6_ILC1 90728796 2109972 2016-01-28 01:00:06 99636027 90728796 2109972 1 2109972 index:0,count:2109972,average:43,stdev:0 GSM1810821_r1 1.84 5.2 0.18 63736976 76784686 47339934 61982728 120.47 130.93 0 0 0 0 0 0 55.24 75.35 3192027 852982 3192027 852982 61.59 71.17 3192027 951136 3192027 805621 8450332 13.26 5.18 0 19.54 0 1.04 0 0.37 0 0.00 0 25.39 0 1544216 0 43 0 41.82 0 1.36 0 0.00 0 1.13 0 0.00 0 281.33 0 0.28 0 109365 0 2109972 0 412234 0 22043 0 7889 0 0 0 535824 0 16 0 0 0 430 0 37432 0 760 0 38638 0 53.65 0 1131982 0 5633 45886 8.145925794426 2109972.0 1544216.0 109365.0 412234.0 22043.0 7889.0 0.0 535824.0 1131982.0 73.2 5.2 19.5 1.0 0.4 0.0 25.4 53.6 43 43 43.00 38 90728796 26.3 22.1 22.0 29.6 0.0 36.2 25.3 smartseq 531518 SRR2088387 SRP060416 SRS980112 SRX1082356 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810822: T75_P4_D7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810822 GSM1810822 T75_P4_D7_ILC1 64396327 1497589 2016-01-28 01:00:06 71206860 64396327 1497589 1 1497589 index:0,count:1497589,average:43,stdev:0 GSM1810822_r1 3.18 5.02 0.2 50384363 64040771 41253540 54724434 127.1 132.65 0 0 0 0 0 0 62.98 77.67 1973191 764504 1973191 764504 67.28 73.6 1973191 816758 1973191 724528 6003884 11.92 3.61 0 15.33 0 1.00 0 0.61 0 0.00 0 17.33 0 1213967 0 43 0 41.91 0 1.34 0 0.00 0 1.21 0 0.00 0 283.75 0 0.28 0 54007 0 1497589 0 229619 0 14915 0 9200 0 0 0 259507 0 23 0 0 0 401 0 38120 0 584 0 39128 0 65.73 0 984348 0 7661 43451 5.671713875473 1497589.0 1213967.0 54007.0 229619.0 14915.0 9200.0 0.0 259507.0 984348.0 81.1 3.6 15.3 1.0 0.6 0.0 17.3 65.7 43 43 43.00 38 64396327 26.1 22.5 22.6 28.8 0.0 36.2 25.5 smartseq 531526 SRR2088388 SRP060416 SRS980109 SRX1082357 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810823: T75_P4_D8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810823 GSM1810823 T75_P4_D8_ILC1 42911764 997948 2016-01-28 01:00:06 47624426 42911764 997948 1 997948 index:0,count:997948,average:43,stdev:0 GSM1810823_r1 2.13 3.65 0.14 36280113 44535905 27747481 36530524 122.76 131.65 0 0 0 0 0 0 58.79 77.38 1742346 511447 1742346 511447 66.35 73.67 1742346 577187 1742346 486896 3920443 10.81 2.36 0 20.94 0 1.36 0 0.57 0 0.00 0 10.90 0 869891 0 43 0 41.98 0 1.37 0 0.00 0 1.12 0 0.00 0 326.60 0 0.28 0 23564 0 997948 0 208960 0 13556 0 5716 0 0 0 108785 0 17 0 0 0 212 0 21020 0 328 0 21577 0 66.23 0 660931 0 5405 26581 4.917853839038 997948.0 869891.0 23564.0 208960.0 13556.0 5716.0 0.0 108785.0 660931.0 87.2 2.4 20.9 1.4 0.6 0.0 10.9 66.2 43 43 43.00 38 42911764 26.7 22.4 22.6 28.3 0.0 36.2 25.7 smartseq 531535 SRR2088389 SRP060416 SRS980110 SRX1082358 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810824: T75_P4_E10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810824 GSM1810824 T75_P4_E10_ILC1 62458274 1452518 2016-01-28 01:00:06 69087130 62458274 1452518 1 1452518 index:0,count:1452518,average:43,stdev:0 GSM1810824_r1 0.99 7.12 0.37 43574286 51562264 35090348 43238340 118.33 123.22 0 0 0 0 0 0 55.49 69.97 1729197 587479 1729197 587479 59.8 66.74 1729197 633037 1729197 560318 7514609 17.25 5.20 0 15.08 0 1.08 0 0.60 0 0.00 0 25.44 0 1058645 0 43 0 41.79 0 1.34 0 0.00 0 1.15 0 0.01 0 249.00 0 0.28 0 75534 0 1452518 0 219057 0 15657 0 8686 0 0 0 369530 0 4 0 0 0 256 0 25664 0 557 0 26481 0 57.80 0 839588 0 4769 29823 6.253512266723 1452518.0 1058645.0 75534.0 219057.0 15657.0 8686.0 0.0 369530.0 839588.0 72.9 5.2 15.1 1.1 0.6 0.0 25.4 57.8 43 43 43.00 38 62458274 26.7 21.5 21.5 30.2 0.0 36.1 25.0 smartseq 531591 SRR2088390 SRP060416 SRS979777 SRX1082359 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810825: T75_P4_E12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810825 GSM1810825 T75_P4_E12_ILC1 158544956 3687092 2016-01-28 01:00:06 173380390 158544956 3687092 1 3687092 index:0,count:3687092,average:43,stdev:0 GSM1810825_r1 1.87 4.72 0.21 119828222 146955190 87929779 116401699 122.64 132.38 0 0 0 0 0 0 57.6 79.33 5967622 1667855 5967622 1667855 66.23 75.37 5967622 1917590 5967622 1584683 12768353 10.66 4.11 0 21.51 0 1.11 0 0.38 0 0.00 0 19.98 0 2895408 0 43 0 41.82 0 1.35 0 0.00 0 1.11 0 0.00 0 323.74 0 0.30 0 151436 0 3687092 0 792913 0 41069 0 13975 0 0 0 736640 0 25 0 0 0 892 0 79649 0 1130 0 81696 0 57.02 0 2102495 0 7399 100402 13.569671577240 3687092.0 2895408.0 151436.0 792913.0 41069.0 13975.0 0.0 736640.0 2102495.0 78.5 4.1 21.5 1.1 0.4 0.0 20.0 57.0 43 43 43.00 38 158544956 26.3 22.3 22.3 29.1 0.0 36.1 25.2 smartseq 531599 SRR2088391 SRP060416 SRS980111 SRX1082360 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810826: T75_P4_E1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810826 GSM1810826 T75_P4_E1_ILC1 169896440 3951080 2016-01-28 01:00:06 185489110 169896440 3951080 1 3951080 index:0,count:3951080,average:43,stdev:0 GSM1810826_r1 1.87 4.14 0.15 128383034 155764546 91643372 122244231 121.33 133.39 0 0 0 0 0 0 55.68 78.75 6944082 1722204 6944082 1722204 64.31 74.7 6944082 1989126 6944082 1633567 14745885 11.49 4.14 0 22.94 0 1.14 0 0.41 0 0.00 0 20.16 0 3093085 0 43 0 41.91 0 1.37 0 0.00 0 1.11 0 0.00 0 296.33 0 0.29 0 163456 0 3951080 0 906291 0 45150 0 16212 0 0 0 796633 0 19 0 0 0 685 0 83693 0 1268 0 85665 0 55.35 0 2186794 0 8672 104750 12.079105166052 3951080.0 3093085.0 163456.0 906291.0 45150.0 16212.0 0.0 796633.0 2186794.0 78.3 4.1 22.9 1.1 0.4 0.0 20.2 55.3 43 43 43.00 38 169896440 26.0 22.5 22.5 28.9 0.0 36.1 25.4 smartseq 531607 SRR2088392 SRP060416 SRS980108 SRX1082361 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810827: T75_P4_E2_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810827 GSM1810827 T75_P4_E2_ILC1 56231358 1307706 2016-01-28 01:00:06 63233840 56231358 1307706 1 1307706 index:0,count:1307706,average:43,stdev:0 GSM1810827_r1 1.94 5.71 0.26 39899247 49952717 29136505 39440320 125.2 135.36 0 0 0 0 0 0 56.37 78.18 1968783 544830 1968783 544830 64.39 73.84 1968783 622356 1968783 514569 4804840 12.04 5.08 0 20.62 0 1.07 0 0.31 0 0.00 0 24.71 0 966551 0 43 0 41.81 0 1.34 0 0.00 0 1.11 0 0.00 0 313.85 0 0.32 0 66436 0 1307706 0 269667 0 13960 0 4024 0 0 0 323171 0 15 0 0 0 283 0 26191 0 449 0 26938 0 53.29 0 696884 0 6321 31770 5.026103464642 1307706.0 966551.0 66436.0 269667.0 13960.0 4024.0 0.0 323171.0 696884.0 73.9 5.1 20.6 1.1 0.3 0.0 24.7 53.3 43 43 43.00 38 56231358 26.0 22.2 22.3 29.5 0.0 35.7 24.5 smartseq 531614 SRR2088393 SRP060416 SRS980107 SRX1082362 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810828: T75_P4_E3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810828 GSM1810828 T75_P4_E3_ILC1 136629834 3177438 2016-01-28 01:00:06 149455107 136629834 3177438 1 3177438 index:0,count:3177438,average:43,stdev:0 GSM1810828_r1 2.25 4.76 0.26 99538162 115775776 76404419 95189431 116.31 124.59 0 0 0 0 0 0 49.43 65.08 4730314 1186003 4730314 1186003 55.44 62.12 4730314 1330353 4730314 1132126 17203348 17.28 4.86 0 18.16 0 1.14 0 0.63 0 0.00 0 22.72 0 2399494 0 43 0 41.92 0 1.35 0 0.00 0 1.14 0 0.00 0 272.35 0 0.29 0 154303 0 3177438 0 577015 0 36309 0 19875 0 0 0 721760 0 11 0 0 0 560 0 50807 0 988 0 52366 0 57.36 0 1822479 0 7424 59546 8.020743534483 3177438.0 2399494.0 154303.0 577015.0 36309.0 19875.0 0.0 721760.0 1822479.0 75.5 4.9 18.2 1.1 0.6 0.0 22.7 57.4 43 43 43.00 38 136629834 26.5 22.1 22.2 29.2 0.0 36.2 25.5 smartseq 531622 SRR2088394 SRP060416 SRS980106 SRX1082363 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810829: T75_P4_E4_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810829 GSM1810829 T75_P4_E4_ILC1 75646976 1759232 2016-01-28 01:00:06 85116399 75646976 1759232 1 1759232 index:0,count:1759232,average:43,stdev:0 GSM1810829_r1 1.85 4.97 0.22 54834773 66450896 40811171 53474981 121.18 131.03 0 0 0 0 0 0 57.0 77.59 2684678 757199 2684678 757199 64.42 73.88 2684678 855750 2684678 720985 6631584 12.09 4.69 0 20.04 0 1.15 0 0.43 0 0.00 0 22.91 0 1328372 0 43 0 41.82 0 1.50 0 0.00 0 1.10 0 0.00 0 211.11 0 0.30 0 82450 0 1759232 0 352525 0 20319 0 7552 0 0 0 402989 0 34 0 0 0 285 0 35461 0 611 0 36391 0 55.47 0 975847 0 6214 43027 6.924203411651 1759232.0 1328372.0 82450.0 352525.0 20319.0 7552.0 0.0 402989.0 975847.0 75.5 4.7 20.0 1.2 0.4 0.0 22.9 55.5 43 43 43.00 38 75646976 26.3 21.9 22.2 29.6 0.0 35.6 24.4 smartseq 531630 SRR2088395 SRP060416 SRS980104 SRX1082364 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810830: T75_P4_E5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810830 GSM1810830 T75_P4_E5_ILC1 135727737 3156459 2016-01-28 01:00:06 148997043 135727737 3156459 1 3156459 index:0,count:3156459,average:43,stdev:0 GSM1810830_r1 1.44 5.39 0.32 93587427 110966317 69822938 89604529 118.57 128.33 0 0 0 0 0 0 54.62 74.29 4617246 1241391 4617246 1241391 61.38 70.93 4617246 1395228 4617246 1185365 13766923 14.71 5.47 0 19.07 0 1.06 0 0.34 0 0.00 0 26.59 0 2272932 0 43 0 41.78 0 1.33 0 0.00 0 1.15 0 0.00 0 291.37 0 0.30 0 172551 0 3156459 0 601842 0 33611 0 10680 0 0 0 839236 0 17 0 0 0 667 0 56568 0 1070 0 58322 0 52.94 0 1671090 0 6448 68615 10.641284119107 3156459.0 2272932.0 172551.0 601842.0 33611.0 10680.0 0.0 839236.0 1671090.0 72.0 5.5 19.1 1.1 0.3 0.0 26.6 52.9 43 43 43.00 38 135727737 26.3 22.0 22.0 29.8 0.0 36.0 24.9 smartseq 531639 SRR2088396 SRP060416 SRS980105 SRX1082365 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810831: T75_P4_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810831 GSM1810831 T75_P4_E6_NK 143691552 3341664 2016-01-28 01:00:06 157236298 143691552 3341664 1 3341664 index:0,count:3341664,average:43,stdev:0 GSM1810831_r1 2.02 4.58 0.21 109287866 133181352 85615570 111460867 121.86 130.19 0 0 0 0 0 0 53.16 68.58 4921485 1399873 4921485 1399873 58.67 65.17 4921485 1545026 4921485 1330281 17070690 15.62 4.01 0 17.72 0 1.25 0 0.64 0 0.00 0 19.31 0 2633409 0 43 0 41.94 0 1.50 0 0.01 0 1.11 0 0.00 0 84.72 0 0.29 0 133872 0 3341664 0 592077 0 41733 0 21355 0 0 0 645167 0 17 0 0 0 595 0 59901 0 1186 0 61699 0 61.09 0 2041332 0 6573 72033 10.958922866271 3341664.0 2633409.0 133872.0 592077.0 41733.0 21355.0 0.0 645167.0 2041332.0 78.8 4.0 17.7 1.2 0.6 0.0 19.3 61.1 43 43 43.00 38 143691552 26.4 22.1 22.1 29.3 0.0 36.2 25.4 smartseq 531647 SRR2088397 SRP060416 SRS980103 SRX1082366 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810832: T75_P4_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810832 GSM1810832 T75_P4_E7_NK 121261161 2820027 2016-01-28 01:00:06 133466708 121261161 2820027 1 2820027 index:0,count:2820027,average:43,stdev:0 GSM1810832_r1 2.67 5.34 0.23 91139519 111772597 72928649 94184337 122.64 129.15 0 0 0 0 0 0 57.73 73.0 3816125 1272320 3816125 1272320 62.77 69.57 3816125 1383349 3816125 1212567 13157244 14.44 4.16 0 16.35 0 1.16 0 0.48 0 0.00 0 20.20 0 2203995 0 43 0 41.84 0 1.39 0 0.00 0 1.16 0 0.00 0 390.47 0 0.29 0 117328 0 2820027 0 461035 0 32828 0 13425 0 0 0 569779 0 21 0 0 0 558 0 57838 0 1015 0 59432 0 61.81 0 1742960 0 7308 67258 9.203338806787 2820027.0 2203995.0 117328.0 461035.0 32828.0 13425.0 0.0 569779.0 1742960.0 78.2 4.2 16.3 1.2 0.5 0.0 20.2 61.8 43 43 43.00 38 121261161 26.4 22.1 22.2 29.3 0.0 36.1 25.1 smartseq 531655 SRR2088398 SRP060416 SRS980101 SRX1082367 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810833: T75_P4_E8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810833 GSM1810833 T75_P4_E8_ILC1 78186771 1818297 2016-01-28 01:00:06 86440591 78186771 1818297 1 1818297 index:0,count:1818297,average:43,stdev:0 GSM1810833_r1 1.65 4.44 0.19 66991003 83571445 51589975 68654514 124.75 133.08 0 0 0 0 0 0 60.04 78.47 3144754 963735 3144754 963735 67.52 74.64 3144754 1083813 3144754 916702 7311190 10.91 2.13 0 20.73 0 1.25 0 0.58 0 0.00 0 9.90 0 1605081 0 43 0 42.01 0 1.41 0 0.00 0 1.16 0 0.00 0 311.71 0 0.29 0 38742 0 1818297 0 376928 0 22646 0 10557 0 0 0 180013 0 22 0 0 0 401 0 42598 0 614 0 43635 0 67.54 0 1228153 0 6814 51605 7.573378338714 1818297.0 1605081.0 38742.0 376928.0 22646.0 10557.0 0.0 180013.0 1228153.0 88.3 2.1 20.7 1.2 0.6 0.0 9.9 67.5 43 43 43.00 38 78186771 26.8 22.3 22.4 28.5 0.0 36.2 25.7 smartseq 532615 SRR2088410 SRP060416 SRS980090 SRX1082379 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810845: T75_P4_G12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810845 GSM1810845 T75_P4_G12_ILC1 142269327 3308589 2016-01-28 01:00:06 154655547 142269327 3308589 1 3308589 index:0,count:3308589,average:43,stdev:0 GSM1810845_r1 2.41 5.74 0.23 102435611 125063751 80400111 103437196 122.09 128.65 0 0 0 0 0 0 59.39 76.68 4460484 1475107 4460484 1475107 65.06 72.81 4460484 1615861 4460484 1400768 13556034 13.23 4.78 0 16.93 0 1.06 0 0.37 0 0.00 0 23.50 0 2483816 0 43 0 41.79 0 1.63 0 0.00 0 1.10 0 0.00 0 313.45 0 0.28 0 158116 0 3308589 0 560057 0 34979 0 12264 0 0 0 777530 0 3 0 0 0 516 0 60997 0 1240 0 62756 0 58.14 0 1923759 0 6077 72039 11.854368932039 3308589.0 2483816.0 158116.0 560057.0 34979.0 12264.0 0.0 777530.0 1923759.0 75.1 4.8 16.9 1.1 0.4 0.0 23.5 58.1 43 43 43.00 38 142269327 26.4 22.0 22.0 29.7 0.0 36.3 25.3 smartseq 532623 SRR2088411 SRP060416 SRS980089 SRX1082380 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810846: T75_P4_G1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810846 GSM1810846 T75_P4_G1_ILC1 141229587 3284409 2016-01-28 01:00:06 153665297 141229587 3284409 1 3284409 index:0,count:3284409,average:43,stdev:0 GSM1810846_r1 2.39 4.26 0.16 107485071 129712624 77957451 102575136 120.68 131.58 0 0 0 0 0 0 54.34 75.66 5653764 1407491 5653764 1407491 63.06 72.24 5653764 1633188 5653764 1343925 13218743 12.30 4.00 0 22.21 0 1.21 0 0.51 0 0.00 0 19.43 0 2589956 0 43 0 41.90 0 1.36 0 0.00 0 1.13 0 0.00 0 337.82 0 0.28 0 131530 0 3284409 0 729598 0 39746 0 16647 0 0 0 638060 0 37 0 0 0 705 0 65458 0 1087 0 67287 0 56.64 0 1860358 0 7765 79835 10.281390856407 3284409.0 2589956.0 131530.0 729598.0 39746.0 16647.0 0.0 638060.0 1860358.0 78.9 4.0 22.2 1.2 0.5 0.0 19.4 56.6 43 43 43.00 38 141229587 26.1 22.4 22.5 28.9 0.0 36.3 25.6 smartseq 532631 SRR2088412 SRP060416 SRS980087 SRX1082381 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810847: T75_P4_G3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810847 GSM1810847 T75_P4_G3_ILC1 127315475 2960825 2016-01-28 01:00:06 138538253 127315475 2960825 1 2960825 index:0,count:2960825,average:43,stdev:0 GSM1810847_r1 1.8 4.0 0.23 95331135 113716393 67950465 90001990 119.29 132.45 0 0 0 0 0 0 53.89 76.33 5282206 1237975 5282206 1237975 62.54 73.02 5282206 1436797 5282206 1184360 11245191 11.80 4.39 0 22.82 0 1.05 0 0.38 0 0.00 0 20.98 0 2297412 0 43 0 41.90 0 1.40 0 0.00 0 1.16 0 0.00 0 380.68 0 0.27 0 130116 0 2960825 0 675536 0 31003 0 11235 0 0 0 621175 0 22 0 0 0 698 0 57187 0 920 0 58827 0 54.78 0 1621876 0 8000 69573 8.696625000000 2960825.0 2297412.0 130116.0 675536.0 31003.0 11235.0 0.0 621175.0 1621876.0 77.6 4.4 22.8 1.0 0.4 0.0 21.0 54.8 43 43 43.00 38 127315475 26.0 22.6 22.6 28.8 0.0 36.3 25.6 smartseq 532639 SRR2088413 SRP060416 SRS980088 SRX1082382 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810848: T75_P4_G5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810848 GSM1810848 T75_P4_G5_ILC1 128804264 2995448 2016-01-28 01:00:06 140561917 128804264 2995448 1 2995448 index:0,count:2995448,average:43,stdev:0 GSM1810848_r1 2.89 4.65 0.2 97991433 122218019 73277853 98923752 124.72 135.0 0 0 0 0 0 0 58.87 79.49 4802290 1389777 4802290 1389777 66.36 75.29 4802290 1566721 4802290 1316376 10327145 10.54 4.11 0 20.44 0 1.04 0 0.41 0 0.00 0 19.73 0 2360828 0 43 0 41.91 0 1.33 0 0.00 0 1.17 0 0.00 0 291.45 0 0.28 0 123146 0 2995448 0 612396 0 31290 0 12406 0 0 0 590924 0 12 0 0 0 657 0 70880 0 979 0 72528 0 58.37 0 1748432 0 8457 83455 9.868156556699 2995448.0 2360828.0 123146.0 612396.0 31290.0 12406.0 0.0 590924.0 1748432.0 78.8 4.1 20.4 1.0 0.4 0.0 19.7 58.4 43 43 43.00 38 128804264 26.1 22.5 22.6 28.8 0.0 36.3 25.5 smartseq 532663 SRR2088416 SRP060416 SRS980084 SRX1082385 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810851: T75_P4_G8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810851 GSM1810851 T75_P4_G8_ILC1 64436317 1498519 2016-01-28 01:00:06 70936418 64436317 1498519 1 1498519 index:0,count:1498519,average:43,stdev:0 GSM1810851_r1 2.75 4.27 0.28 51769191 63396276 41141760 53066839 122.46 128.99 0 0 0 0 0 0 60.66 77.08 2242841 757588 2242841 757588 66.31 73.71 2242841 828251 2242841 724458 6615824 12.78 3.20 0 17.76 0 1.12 0 0.60 0 0.00 0 14.93 0 1249009 0 43 0 41.86 0 1.48 0 0.00 0 1.15 0 0.00 0 414.97 0 0.28 0 47906 0 1498519 0 266140 0 16832 0 9009 0 0 0 223669 0 4 0 0 0 501 0 31564 0 550 0 32619 0 65.59 0 982869 0 4201 36873 8.777195905737 1498519.0 1249009.0 47906.0 266140.0 16832.0 9009.0 0.0 223669.0 982869.0 83.3 3.2 17.8 1.1 0.6 0.0 14.9 65.6 43 43 43.00 38 64436317 26.9 22.0 22.1 29.0 0.0 36.3 25.4 smartseq 532671 SRR2088417 SRP060416 SRS980083 SRX1082386 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1810852: T75_P4_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T75 GEO Accession;;GSM1810852 GSM1810852 T75_P4_G9_NK 26805684 623388 2016-01-28 01:00:06 29634427 26805684 623388 1 623388 index:0,count:623388,average:43,stdev:0 GSM1810852_r1 3.3 5.19 0.19 20138297 24659920 16077742 20797867 122.45 129.36 0 0 0 0 0 0 56.35 71.38 857393 273902 857393 273902 61.88 68.5 857393 300776 857393 262829 3161436 15.70 4.27 0 16.42 0 1.07 0 0.55 0 0.00 0 20.41 0 486066 0 43 0 41.90 0 1.47 0 0.00 0 1.15 0 0.00 0 132.01 0 0.27 0 26589 0 623388 0 102355 0 6684 0 3419 0 0 0 127219 0 9 0 0 0 104 0 10832 0 237 0 11182 0 61.55 0 383711 0 4003 12516 3.126655008743 623388.0 486066.0 26589.0 102355.0 6684.0 3419.0 0.0 127219.0 383711.0 78.0 4.3 16.4 1.1 0.5 0.0 20.4 61.6 43 43 43.00 38 26805684 26.7 21.9 21.9 29.4 0.0 36.3 25.5 smartseq 536727 SRR2088612 SRP060416 SRS979994 SRX1082581 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811053: T86_P3_E3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811053 GSM1811053 T86_P3_E3_ILC1 134432534 3126338 2016-01-28 01:00:06 144471989 134432534 3126338 1 3126338 index:0,count:3126338,average:43,stdev:0 GSM1811053_r1 7.49 6.28 0.18 82048662 107172909 61882314 84282378 130.62 136.2 0 0 0 0 0 0 57.84 77.79 3489642 1155343 3489642 1155343 67.6 74.12 3489642 1350182 3489642 1100848 10303434 12.56 7.80 0 16.38 0 1.00 0 0.35 0 0.00 0 34.76 0 1997386 0 43 0 41.67 0 1.13 0 0.01 0 1.14 0 0.01 0 281.37 0 0.33 0 243720 0 3126338 0 512231 0 31135 0 11022 0 0 0 1086795 0 6 0 0 0 323 0 49088 0 981 0 50398 0 47.50 0 1485155 0 8984 62079 6.909951024043 3126338.0 1997386.0 243720.0 512231.0 31135.0 11022.0 0.0 1086795.0 1485155.0 63.9 7.8 16.4 1.0 0.4 0.0 34.8 47.5 43 43 43.00 38 134432534 26.1 22.6 22.6 28.8 0.0 36.3 24.8 smartseq 536735 SRR2088613 SRP060416 SRS979993 SRX1082582 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811054: T86_P3_E5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811054 GSM1811054 T86_P3_E5_ILC1 127356282 2961774 2016-01-28 01:00:06 137336637 127356282 2961774 1 2961774 index:0,count:2961774,average:43,stdev:0 GSM1811054_r1 5.69 6.84 0.18 78472167 100568580 62655665 83346269 128.16 133.02 0 0 0 0 0 0 61.04 77.63 3067632 1166071 3067632 1166071 67.25 74.21 3067632 1284638 3067632 1114736 10493384 13.37 7.65 0 13.78 0 0.89 0 0.33 0 0.00 0 34.29 0 1910339 0 43 0 41.71 0 1.14 0 0.01 0 1.08 0 0.01 0 226.86 0 0.33 0 226681 0 2961774 0 408203 0 26321 0 9666 0 0 0 1015448 0 3 0 0 0 492 0 53576 0 890 0 54961 0 50.72 0 1502136 0 9032 62319 6.899800708592 2961774.0 1910339.0 226681.0 408203.0 26321.0 9666.0 0.0 1015448.0 1502136.0 64.5 7.7 13.8 0.9 0.3 0.0 34.3 50.7 43 43 43.00 38 127356282 26.1 22.5 22.6 28.8 0.0 36.2 24.7 smartseq 536742 SRR2088614 SRP060416 SRS979995 SRX1082583 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811055: T86_P3_E6_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811055 GSM1811055 T86_P3_E6_NK 158186551 3678757 2016-01-28 01:00:06 169775939 158186551 3678757 1 3678757 index:0,count:3678757,average:43,stdev:0 GSM1811055_r1 6.04 5.78 0.18 109165929 142633702 89763899 120903356 130.66 134.69 0 0 0 0 0 0 59.46 73.14 4014729 1569655 4014729 1569655 65.08 69.76 4014729 1718127 4014729 1497134 14637598 13.41 5.96 0 13.43 0 1.09 0 0.42 0 0.00 0 26.73 0 2640007 0 43 0 41.83 0 1.19 0 0.01 0 1.11 0 0.00 0 275.91 0 0.34 0 219275 0 3678757 0 493996 0 40155 0 15424 0 0 0 983171 0 52 0 0 0 529 0 67811 0 1265 0 69657 0 58.34 0 2146011 0 10576 79679 7.533944780635 3678757.0 2640007.0 219275.0 493996.0 40155.0 15424.0 0.0 983171.0 2146011.0 71.8 6.0 13.4 1.1 0.4 0.0 26.7 58.3 43 43 43.00 38 158186551 25.7 23.1 23.1 28.0 0.0 36.3 24.9 smartseq 536750 SRR2088615 SRP060416 SRS979992 SRX1082584 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811056: T86_P3_E7_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811056 GSM1811056 T86_P3_E7_NK 147259821 3424647 2016-01-28 01:00:06 158222322 147259821 3424647 1 3424647 index:0,count:3424647,average:43,stdev:0 GSM1811056_r1 9.65 5.84 0.16 103429656 139723589 83116640 114995111 135.09 138.35 0 0 0 0 0 0 64.21 80.72 3867616 1602601 3867616 1602601 72.34 77.23 3867616 1805594 3867616 1533399 10970331 10.61 5.77 0 14.91 0 0.92 0 0.34 0 0.00 0 25.86 0 2496038 0 43 0 41.86 0 1.15 0 0.01 0 1.12 0 0.01 0 251.61 0 0.32 0 197732 0 3424647 0 510619 0 31558 0 11499 0 0 0 885552 0 34 0 0 0 584 0 68798 0 1136 0 70552 0 57.97 0 1985419 0 11747 82590 7.030731250532 3424647.0 2496038.0 197732.0 510619.0 31558.0 11499.0 0.0 885552.0 1985419.0 72.9 5.8 14.9 0.9 0.3 0.0 25.9 58.0 43 43 43.00 38 147259821 26.0 22.9 22.9 28.3 0.0 36.3 25.1 smartseq 536758 SRR2088616 SRP060416 SRS979991 SRX1082585 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811057: T86_P3_E8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811057 GSM1811057 T86_P3_E8_ILC1 87688266 2039262 2016-01-28 01:00:06 95110580 87688266 2039262 1 2039262 index:0,count:2039262,average:43,stdev:0 GSM1811057_r1 6.85 6.59 0.17 54108506 70984115 42095281 57312567 131.19 136.15 0 0 0 0 0 0 60.25 78.53 2165800 791559 2165800 791559 68.63 75.03 2165800 901614 2165800 756212 6856740 12.67 7.65 0 15.00 0 0.91 0 0.37 0 0.00 0 34.29 0 1313722 0 43 0 41.76 0 1.14 0 0.01 0 1.15 0 0.01 0 198.41 0 0.33 0 156096 0 2039262 0 305809 0 18543 0 7634 0 0 0 699363 0 6 0 0 0 246 0 32670 0 641 0 33563 0 49.43 0 1007913 0 7299 38996 5.342649678038 2039262.0 1313722.0 156096.0 305809.0 18543.0 7634.0 0.0 699363.0 1007913.0 64.4 7.7 15.0 0.9 0.4 0.0 34.3 49.4 43 43 43.00 38 87688266 25.8 22.8 22.8 28.6 0.0 36.2 24.6 smartseq 536767 SRR2088617 SRP060416 SRS979989 SRX1082586 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811058: T86_P3_E9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811058 GSM1811058 T86_P3_E9_ILC1 25568273 594611 2016-01-28 01:00:06 27930942 25568273 594611 1 594611 index:0,count:594611,average:43,stdev:0 GSM1811058_r1 7.55 6.27 0.19 15708559 20656034 12077477 16535586 131.5 136.91 0 0 0 0 0 0 59.4 78.34 651186 226652 651186 226652 68.47 74.95 651186 261254 651186 216826 1878704 11.96 7.74 0 15.52 0 1.01 0 0.31 0 0.00 0 34.51 0 381576 0 43 0 41.75 0 1.16 0 0.01 0 1.09 0 0.00 0 101.93 0 0.33 0 46024 0 594611 0 92263 0 6001 0 1842 0 0 0 205192 0 1 0 0 0 56 0 9412 0 191 0 9660 0 48.66 0 289313 0 4380 10868 2.481278538813 594611.0 381576.0 46024.0 92263.0 6001.0 1842.0 0.0 205192.0 289313.0 64.2 7.7 15.5 1.0 0.3 0.0 34.5 48.7 43 43 43.00 38 25568273 25.9 22.8 22.8 28.5 0.0 36.2 24.7 smartseq 536774 SRR2088618 SRP060416 SRS979990 SRX1082587 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811059: T86_P3_F11_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811059 GSM1811059 T86_P3_F11_ILC1 132384960 3078720 2016-01-28 01:00:06 142612961 132384960 3078720 1 3078720 index:0,count:3078720,average:43,stdev:0 GSM1811059_r1 7.16 7.67 0.19 80472986 104713774 62415050 84303601 130.12 135.07 0 0 0 0 0 0 60.87 79.82 3246943 1197816 3246943 1197816 68.86 76.51 3246943 1354938 3246943 1148277 10227045 12.71 7.82 0 15.17 0 0.94 0 0.28 0 0.00 0 34.87 0 1967769 0 43 0 41.59 0 1.13 0 0.01 0 1.13 0 0.00 0 230.90 0 0.35 0 240793 0 3078720 0 467032 0 28843 0 8594 0 0 0 1073514 0 10 0 0 0 386 0 49770 0 1029 0 51195 0 48.75 0 1500737 0 8370 58886 7.035364396655 3078720.0 1967769.0 240793.0 467032.0 28843.0 8594.0 0.0 1073514.0 1500737.0 63.9 7.8 15.2 0.9 0.3 0.0 34.9 48.7 43 43 43.00 38 132384960 26.0 22.5 22.4 29.1 0.0 36.2 24.5 smartseq 536783 SRR2088619 SRP060416 SRS979887 SRX1082588 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811060: T86_P3_F12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811060 GSM1811060 T86_P3_F12_ILC1 169473922 3941254 2016-01-28 01:00:06 181674867 169473922 3941254 1 3941254 index:0,count:3941254,average:43,stdev:0 GSM1811060_r1 7.16 6.7 0.2 107996761 143381634 84054768 115794267 132.76 137.76 0 0 0 0 0 0 61.16 79.75 4354204 1608349 4354204 1608349 69.51 76.17 4354204 1828009 4354204 1536154 12861034 11.91 7.17 0 15.56 0 0.93 0 0.31 0 0.00 0 32.04 0 2629921 0 43 0 41.68 0 1.12 0 0.01 0 1.10 0 0.00 0 267.71 0 0.34 0 282574 0 3941254 0 613253 0 36667 0 12049 0 0 0 1262617 0 16 0 0 0 501 0 68152 0 1460 0 70129 0 51.17 0 2016668 0 9360 82081 8.769337606838 3941254.0 2629921.0 282574.0 613253.0 36667.0 12049.0 0.0 1262617.0 2016668.0 66.7 7.2 15.6 0.9 0.3 0.0 32.0 51.2 43 43 43.00 38 169473922 25.9 22.7 22.7 28.7 0.0 36.2 24.7 smartseq 536838 SRR2088620 SRP060416 SRS979885 SRX1082589 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811061: T86_P3_F1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811061 GSM1811061 T86_P3_F1_ILC1 178719438 4156266 2016-01-28 01:00:06 191386505 178719438 4156266 1 4156266 index:0,count:4156266,average:43,stdev:0 GSM1811061_r1 7.44 6.42 0.16 117733530 157053541 92197363 127633491 133.4 138.44 0 0 0 0 0 0 62.57 80.94 4673064 1786267 4673064 1786267 70.66 76.89 4673064 2017243 4673064 1696844 13239635 11.25 6.70 0 15.59 0 0.85 0 0.35 0 0.00 0 30.11 0 2855022 0 43 0 41.78 0 1.14 0 0.01 0 1.10 0 0.00 0 277.08 0 0.34 0 278495 0 4156266 0 648119 0 35389 0 14543 0 0 0 1251312 0 35 0 0 0 713 0 82950 0 1418 0 85116 0 53.10 0 2206903 0 11666 100425 8.608349048517 4156266.0 2855022.0 278495.0 648119.0 35389.0 14543.0 0.0 1251312.0 2206903.0 68.7 6.7 15.6 0.9 0.3 0.0 30.1 53.1 43 43 43.00 38 178719438 25.9 22.8 22.8 28.5 0.0 36.2 24.7 smartseq 536847 SRR2088621 SRP060416 SRS979886 SRX1082590 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811062: T86_P3_F3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811062 GSM1811062 T86_P3_F3_ILC1 149550689 3477923 2016-01-28 01:00:06 160839321 149550689 3477923 1 3477923 index:0,count:3477923,average:43,stdev:0 GSM1811062_r1 3.16 6.35 0.19 99316349 123723131 78647854 100638792 124.57 127.96 0 0 0 0 0 0 58.14 74.36 3900702 1400221 3900702 1400221 65.56 70.54 3900702 1578698 3900702 1328384 14335760 14.43 6.55 0 15.10 0 1.02 0 0.45 0 0.00 0 29.29 0 2408191 0 43 0 41.76 0 1.24 0 0.01 0 1.11 0 0.00 0 240.78 0 0.34 0 227794 0 3477923 0 525080 0 35384 0 15497 0 0 0 1018851 0 20 0 0 0 640 0 70097 0 1232 0 71989 0 54.14 0 1883111 0 11852 86170 7.270502868714 3477923.0 2408191.0 227794.0 525080.0 35384.0 15497.0 0.0 1018851.0 1883111.0 69.2 6.5 15.1 1.0 0.4 0.0 29.3 54.1 43 43 43.00 38 149550689 26.0 22.7 22.7 28.6 0.0 36.2 24.8 smartseq 536854 SRR2088622 SRP060416 SRS979988 SRX1082591 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811063: T86_P3_F4_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811063 GSM1811063 T86_P3_F4_ILC1 101179215 2353005 2016-01-28 01:00:06 109437794 101179215 2353005 1 2353005 index:0,count:2353005,average:43,stdev:0 GSM1811063_r1 8.66 6.22 0.17 67692673 91396605 54113179 75325635 135.02 139.2 0 0 0 0 0 0 62.96 79.7 2556672 1031600 2556672 1031600 70.8 76.32 2556672 1160004 2556672 987830 7750593 11.45 6.52 0 14.62 0 0.83 0 0.30 0 0.00 0 29.25 0 1638399 0 43 0 41.81 0 1.20 0 0.01 0 1.13 0 0.01 0 282.36 0 0.33 0 153309 0 2353005 0 344021 0 19417 0 6993 0 0 0 688196 0 19 0 0 0 397 0 45559 0 860 0 46835 0 55.01 0 1294378 0 10305 53509 5.192527899078 2353005.0 1638399.0 153309.0 344021.0 19417.0 6993.0 0.0 688196.0 1294378.0 69.6 6.5 14.6 0.8 0.3 0.0 29.2 55.0 43 43 43.00 38 101179215 25.9 22.8 22.9 28.4 0.0 36.3 24.8 smartseq 536863 SRR2088623 SRP060416 SRS979987 SRX1082592 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811064: T86_P3_F5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811064 GSM1811064 T86_P3_F5_ILC1 153766065 3575955 2016-01-28 01:00:06 165389163 153766065 3575955 1 3575955 index:0,count:3575955,average:43,stdev:0 GSM1811064_r1 8.4 6.21 0.15 103320601 138179745 82143658 113104985 133.74 137.69 0 0 0 0 0 0 63.31 80.58 3936426 1584351 3936426 1584351 71.56 76.88 3936426 1790763 3936426 1511545 11227178 10.87 6.42 0 15.00 0 0.87 0 0.36 0 0.00 0 28.80 0 2502382 0 43 0 41.78 0 1.14 0 0.01 0 1.13 0 0.00 0 238.40 0 0.33 0 229434 0 3575955 0 536233 0 30977 0 12713 0 0 0 1029883 0 20 0 0 0 545 0 71417 0 1183 0 73165 0 54.98 0 1966149 0 10467 86482 8.262348332856 3575955.0 2502382.0 229434.0 536233.0 30977.0 12713.0 0.0 1029883.0 1966149.0 70.0 6.4 15.0 0.9 0.4 0.0 28.8 55.0 43 43 43.00 38 153766065 26.0 22.9 23.0 28.2 0.0 36.2 24.8 smartseq 536871 SRR2088624 SRP060416 SRS979986 SRX1082593 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811065: T86_P3_F6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811065 GSM1811065 T86_P3_F6_ILC1 170180756 3957692 2016-01-28 01:00:06 183280522 170180756 3957692 1 3957692 index:0,count:3957692,average:43,stdev:0 GSM1811065_r1 8.9 6.02 0.27 111027958 148582177 84922543 118512408 133.82 139.55 0 0 0 0 0 0 58.49 77.5 4590834 1578591 4590834 1578591 68.14 74.35 4590834 1839164 4590834 1514325 14264147 12.85 6.79 0 16.73 0 0.97 0 0.39 0 0.00 0 30.44 0 2698913 0 43 0 41.69 0 1.12 0 0.01 0 1.13 0 0.01 0 309.73 0 0.35 0 268607 0 3957692 0 662093 0 38487 0 15584 0 0 0 1204708 0 40 0 0 0 415 0 61594 0 1219 0 63268 0 51.46 0 2036820 0 8061 75715 9.392755241285 3957692.0 2698913.0 268607.0 662093.0 38487.0 15584.0 0.0 1204708.0 2036820.0 68.2 6.8 16.7 1.0 0.4 0.0 30.4 51.5 43 43 43.00 38 170180756 26.1 22.7 22.7 28.5 0.0 36.1 24.6 smartseq 536879 SRR2088625 SRP060416 SRS979985 SRX1082594 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811066: T86_P3_F7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811066 GSM1811066 T86_P3_F7_ILC1 120822389 2809823 2016-01-28 01:00:06 130445758 120822389 2809823 1 2809823 index:0,count:2809823,average:43,stdev:0 GSM1811066_r1 3.26 7.8 0.25 71133910 87509824 54267343 69680059 123.02 128.4 0 0 0 0 0 0 54.71 73.14 3003133 956014 3003133 956014 62.42 70.04 3003133 1090757 3003133 915555 11248214 15.81 8.15 0 15.67 0 1.06 0 0.29 0 0.00 0 36.46 0 1747526 0 43 0 41.52 0 1.22 0 0.01 0 1.14 0 0.00 0 224.79 0 0.34 0 229032 0 2809823 0 440364 0 29668 0 8074 0 0 0 1024555 0 15 0 0 0 308 0 41532 0 1058 0 42913 0 46.52 0 1307162 0 7257 51309 7.070276973956 2809823.0 1747526.0 229032.0 440364.0 29668.0 8074.0 0.0 1024555.0 1307162.0 62.2 8.2 15.7 1.1 0.3 0.0 36.5 46.5 43 43 43.00 38 120822389 26.2 22.2 22.1 29.5 0.0 36.1 24.3 smartseq 536886 SRR2088626 SRP060416 SRS979984 SRX1082595 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811067: T86_P3_F8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811067 GSM1811067 T86_P3_F8_ILC1 77594661 1804527 2016-01-28 01:00:06 84378276 77594661 1804527 1 1804527 index:0,count:1804527,average:43,stdev:0 GSM1811067_r1 8.31 6.51 0.17 51470600 69830450 40011674 56181113 135.67 140.41 0 0 0 0 0 0 61.31 79.84 2043559 764243 2043559 764243 70.82 76.26 2043559 882787 2043559 729928 5968670 11.60 6.63 0 16.03 0 0.92 0 0.33 0 0.00 0 29.68 0 1246560 0 43 0 41.80 0 1.13 0 0.01 0 1.11 0 0.00 0 196.86 0 0.34 0 119644 0 1804527 0 289346 0 16542 0 5926 0 0 0 535499 0 12 0 0 0 193 0 32859 0 519 0 33583 0 53.05 0 957214 0 8173 39545 4.838492597577 1804527.0 1246560.0 119644.0 289346.0 16542.0 5926.0 0.0 535499.0 957214.0 69.1 6.6 16.0 0.9 0.3 0.0 29.7 53.0 43 43 43.00 38 77594661 25.9 22.8 22.8 28.4 0.0 36.2 24.7 smartseq 536895 SRR2088627 SRP060416 SRS979983 SRX1082596 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811068: T86_P3_G10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811068 GSM1811068 T86_P3_G10_ILC1 54597659 1269713 2016-01-28 01:00:06 59350171 54597659 1269713 1 1269713 index:0,count:1269713,average:43,stdev:0 GSM1811068_r1 8.99 6.2 0.16 35609379 48021135 27626235 38448226 134.86 139.17 0 0 0 0 0 0 62.1 81.03 1412266 535686 1412266 535686 72.23 77.86 1412266 623069 1412266 514722 3967865 11.14 6.95 0 15.87 0 0.91 0 0.27 0 0.00 0 30.88 0 862629 0 43 0 41.79 0 1.14 0 0.01 0 1.11 0 0.01 0 240.58 0 0.33 0 88234 0 1269713 0 201514 0 11555 0 3430 0 0 0 392099 0 0 0 0 0 138 0 23284 0 360 0 23782 0 52.07 0 661115 0 7063 28054 3.971966586436 1269713.0 862629.0 88234.0 201514.0 11555.0 3430.0 0.0 392099.0 661115.0 67.9 6.9 15.9 0.9 0.3 0.0 30.9 52.1 43 43 43.00 38 54597659 26.0 22.7 22.8 28.5 0.0 36.3 24.9 smartseq 536902 SRR2088628 SRP060416 SRS979981 SRX1082597 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811069: T86_P3_G12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811069 GSM1811069 T86_P3_G12_ILC1 148905302 3462914 2016-01-28 01:00:06 159668393 148905302 3462914 1 3462914 index:0,count:3462914,average:43,stdev:0 GSM1811069_r1 7.47 7.62 0.24 81852699 102723190 60581502 79494222 125.5 131.22 0 0 0 0 0 0 52.75 72.77 3576036 1064696 3576036 1064696 61.73 69.79 3576036 1245997 3576036 1021118 13478545 16.47 9.02 0 16.04 0 1.06 0 0.28 0 0.00 0 40.37 0 2018488 0 43 0 41.40 0 1.11 0 0.01 0 1.14 0 0.01 0 259.72 0 0.36 0 312345 0 3462914 0 555327 0 36826 0 9790 0 0 0 1397810 0 60 0 0 0 354 0 38963 0 1217 0 40594 0 42.25 0 1463161 0 6168 47898 7.765564202335 3462914.0 2018488.0 312345.0 555327.0 36826.0 9790.0 0.0 1397810.0 1463161.0 58.3 9.0 16.0 1.1 0.3 0.0 40.4 42.3 43 43 43.00 38 148905302 26.1 22.2 22.2 29.5 0.0 36.2 24.3 smartseq 536910 SRR2088629 SRP060416 SRS979980 SRX1082598 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811070: T86_P3_G1_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811070 GSM1811070 T86_P3_G1_ILC1 190765028 4436396 2016-01-28 01:00:06 203363435 190765028 4436396 1 4436396 index:0,count:4436396,average:43,stdev:0 GSM1811070_r1 8.26 5.93 0.2 132824378 179591498 103717766 144588942 135.21 139.41 0 0 0 0 0 0 63.35 82.02 5188008 2032829 5188008 2032829 72.69 78.04 5188008 2332655 5188008 1934255 14013637 10.55 5.88 0 16.47 0 0.94 0 0.31 0 0.00 0 26.41 0 3209049 0 43 0 41.85 0 1.13 0 0.01 0 1.12 0 0.00 0 280.19 0 0.33 0 261044 0 4436396 0 730619 0 41792 0 13874 0 0 0 1171681 0 33 0 0 0 735 0 93808 0 1508 0 96084 0 55.87 0 2478430 0 12321 114393 9.284392500609 4436396.0 3209049.0 261044.0 730619.0 41792.0 13874.0 0.0 1171681.0 2478430.0 72.3 5.9 16.5 0.9 0.3 0.0 26.4 55.9 43 43 43.00 38 190765028 25.8 22.9 23.0 28.2 0.0 36.4 25.0 smartseq 536966 SRR2088630 SRP060416 SRS979888 SRX1082599 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811071: T86_P3_G3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811071 GSM1811071 T86_P3_G3_ILC1 135386231 3148517 2016-01-28 01:00:06 144928003 135386231 3148517 1 3148517 index:0,count:3148517,average:43,stdev:0 GSM1811071_r1 8.59 6.02 0.17 93109430 125379592 72245189 100352992 134.66 138.91 0 0 0 0 0 0 62.24 81.18 3713179 1405145 3713179 1405145 72.1 77.67 3713179 1627800 3713179 1344304 9919499 10.65 6.01 0 16.73 0 0.99 0 0.28 0 0.00 0 27.02 0 2257713 0 43 0 41.74 0 1.13 0 0.01 0 1.12 0 0.00 0 298.28 0 0.33 0 189197 0 3148517 0 526813 0 31252 0 8875 0 0 0 850677 0 15 0 0 0 420 0 63545 0 1068 0 65048 0 54.98 0 1730900 0 10887 77290 7.099292734454 3148517.0 2257713.0 189197.0 526813.0 31252.0 8875.0 0.0 850677.0 1730900.0 71.7 6.0 16.7 1.0 0.3 0.0 27.0 55.0 43 43 43.00 38 135386231 26.1 22.7 22.7 28.5 0.0 36.4 25.0 smartseq 536974 SRR2088631 SRP060416 SRS979979 SRX1082600 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811072: T86_P3_G5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811072 GSM1811072 T86_P3_G5_ILC1 149079452 3466964 2016-01-28 01:00:06 159919099 149079452 3466964 1 3466964 index:0,count:3466964,average:43,stdev:0 GSM1811072_r1 8.2 6.2 0.2 100848039 135279664 80190127 110842830 134.14 138.23 0 0 0 0 0 0 62.68 79.75 3879969 1529396 3879969 1529396 70.39 75.93 3879969 1717590 3879969 1456212 11623926 11.53 6.37 0 15.06 0 0.85 0 0.31 0 0.00 0 28.46 0 2439999 0 43 0 41.81 0 1.12 0 0.01 0 1.12 0 0.01 0 312.03 0 0.33 0 220713 0 3466964 0 522222 0 29422 0 10732 0 0 0 986811 0 27 0 0 0 515 0 66548 0 1183 0 68273 0 55.32 0 1917777 0 10447 78974 7.559490762898 3466964.0 2439999.0 220713.0 522222.0 29422.0 10732.0 0.0 986811.0 1917777.0 70.4 6.4 15.1 0.8 0.3 0.0 28.5 55.3 43 43 43.00 38 149079452 26.1 22.8 22.8 28.3 0.0 36.3 24.9 smartseq 536983 SRR2088632 SRP060416 SRS979982 SRX1082601 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811073: T86_P3_G6_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811073 GSM1811073 T86_P3_G6_ILC1 160071456 3722592 2016-01-28 01:00:06 172833889 160071456 3722592 1 3722592 index:0,count:3722592,average:43,stdev:0 GSM1811073_r1 9.7 5.91 0.19 110179705 149914090 85401032 120427256 136.06 141.01 0 0 0 0 0 0 61.16 79.81 4446243 1630848 4446243 1630848 70.93 76.4 4446243 1891203 4446243 1561215 12467338 11.32 6.01 0 16.73 0 0.95 0 0.35 0 0.00 0 27.07 0 2666406 0 43 0 41.79 0 1.16 0 0.01 0 1.13 0 0.00 0 252.86 0 0.35 0 223891 0 3722592 0 622865 0 35317 0 13211 0 0 0 1007658 0 24 0 0 0 514 0 67741 0 1189 0 69468 0 54.90 0 2043541 0 9569 81307 8.496917128227 3722592.0 2666406.0 223891.0 622865.0 35317.0 13211.0 0.0 1007658.0 2043541.0 71.6 6.0 16.7 0.9 0.4 0.0 27.1 54.9 43 43 43.00 38 160071456 25.9 22.9 22.9 28.3 0.0 36.1 24.7 smartseq 536990 SRR2088633 SRP060416 SRS979978 SRX1082602 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811074: T86_P3_G7_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811074 GSM1811074 T86_P3_G7_ILC1 108514112 2523584 2016-01-28 01:00:06 117835822 108514112 2523584 1 2523584 index:0,count:2523584,average:43,stdev:0 GSM1811074_r1 8.78 6.21 0.19 73321531 98718973 56527476 78735910 134.64 139.29 0 0 0 0 0 0 60.97 80.12 2925299 1085389 2925299 1085389 70.86 76.7 2925299 1261499 2925299 1039136 8253728 11.26 6.27 0 16.86 0 0.93 0 0.30 0 0.00 0 28.22 0 1780340 0 43 0 41.72 0 1.17 0 0.01 0 1.11 0 0.01 0 267.20 0 0.34 0 158247 0 2523584 0 425571 0 23433 0 7618 0 0 0 712193 0 26 0 0 0 355 0 44298 0 942 0 45621 0 53.68 0 1354769 0 8752 53345 6.095178244973 2523584.0 1780340.0 158247.0 425571.0 23433.0 7618.0 0.0 712193.0 1354769.0 70.5 6.3 16.9 0.9 0.3 0.0 28.2 53.7 43 43 43.00 38 108514112 26.2 22.5 22.6 28.7 0.0 36.1 24.6 smartseq 536998 SRR2088634 SRP060416 SRS979977 SRX1082603 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811075: T86_P3_G8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811075 GSM1811075 T86_P3_G8_ILC1 81882793 1904251 2016-01-28 01:00:06 88656395 81882793 1904251 1 1904251 index:0,count:1904251,average:43,stdev:0 GSM1811075_r1 9.88 6.2 0.19 55179361 75978969 43763370 62014714 137.69 141.7 0 0 0 0 0 0 65.11 82.97 2081571 867575 2081571 867575 74.01 79.48 2081571 986118 2081571 831127 5486618 9.94 6.47 0 15.06 0 0.80 0 0.28 0 0.00 0 28.95 0 1332394 0 43 0 41.85 0 1.13 0 0.01 0 1.12 0 0.00 0 214.23 0 0.33 0 123230 0 1904251 0 286733 0 15180 0 5368 0 0 0 551309 0 38 0 0 0 335 0 37295 0 604 0 38272 0 54.91 0 1045661 0 9554 44168 4.622985137115 1904251.0 1332394.0 123230.0 286733.0 15180.0 5368.0 0.0 551309.0 1045661.0 70.0 6.5 15.1 0.8 0.3 0.0 29.0 54.9 43 43 43.00 38 81882793 25.8 23.0 23.0 28.1 0.0 36.3 25.0 smartseq 537006 SRR2088635 SRP060416 SRS979976 SRX1082604 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811076: T86_P3_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811076 GSM1811076 T86_P3_G9_NK 31042861 721927 2016-01-28 01:00:06 33760330 31042861 721927 1 721927 index:0,count:721927,average:43,stdev:0 GSM1811076_r1 9.79 5.81 0.16 20931112 28259735 16644973 23195768 135.01 139.36 0 0 0 0 0 0 60.99 77.6 797391 308671 797391 308671 69.39 74.38 797391 351167 797391 295863 2561955 12.24 6.39 0 15.00 0 0.93 0 0.41 0 0.00 0 28.56 0 506067 0 43 0 41.85 0 1.14 0 0.01 0 1.09 0 0.00 0 162.43 0 0.32 0 46121 0 721927 0 108298 0 6731 0 2946 0 0 0 206183 0 5 0 0 0 98 0 12372 0 229 0 12704 0 55.10 0 397769 0 5517 14730 2.669929309407 721927.0 506067.0 46121.0 108298.0 6731.0 2946.0 0.0 206183.0 397769.0 70.1 6.4 15.0 0.9 0.4 0.0 28.6 55.1 43 43 43.00 38 31042861 26.1 22.8 22.8 28.4 0.0 36.4 25.0 smartseq 537014 SRR2088636 SRP060416 SRS979974 SRX1082605 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811077: T86_P3_H10_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811077 GSM1811077 T86_P3_H10_ILC1 26497116 616212 2016-01-28 01:00:06 30372584 26497116 616212 1 616212 index:0,count:616212,average:43,stdev:0 GSM1811077_r1 8.28 6.8 0.22 17881840 23969428 13627248 18898240 134.04 138.68 0 0 0 0 0 0 61.6 81.83 726314 267278 726314 267278 71.86 77.97 726314 311822 726314 254648 1885637 10.54 6.41 0 17.41 0 0.94 0 0.25 0 0.00 0 28.40 0 433903 0 43 0 41.72 0 1.14 0 0.01 0 1.13 0 0.00 0 110.92 0 0.45 0 39500 0 616212 0 107296 0 5796 0 1515 0 0 0 174998 0 6 0 0 0 108 0 12044 0 195 0 12353 0 53.00 0 326607 0 5906 14475 2.450897392482 616212.0 433903.0 39500.0 107296.0 5796.0 1515.0 0.0 174998.0 326607.0 70.4 6.4 17.4 0.9 0.2 0.0 28.4 53.0 43 43 43.00 38 26497116 26.2 22.2 23.3 28.4 0.0 34.9 23.1 smartseq 537031 SRR2088638 SRP060416 SRS979973 SRX1082607 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811079: T86_P3_H12_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811079 GSM1811079 T86_P3_H12_ILC1 113621652 2642364 2016-01-28 01:00:06 123216771 113621652 2642364 1 2642364 index:0,count:2642364,average:43,stdev:0 GSM1811079_r1 9.22 6.43 0.18 72675031 97613206 54776815 76445808 134.31 139.56 0 0 0 0 0 0 61.25 82.45 3030647 1085910 3030647 1085910 71.95 78.94 3030647 1275667 3030647 1039696 7857550 10.81 7.12 0 17.25 0 0.94 0 0.23 0 0.00 0 31.73 0 1773013 0 43 0 41.59 0 1.10 0 0.01 0 1.14 0 0.00 0 352.32 0 0.35 0 188011 0 2642364 0 455896 0 24901 0 6050 0 0 0 838400 0 25 0 0 0 349 0 46867 0 818 0 48059 0 49.85 0 1317117 0 9580 57566 6.008977035491 2642364.0 1773013.0 188011.0 455896.0 24901.0 6050.0 0.0 838400.0 1317117.0 67.1 7.1 17.3 0.9 0.2 0.0 31.7 49.8 43 43 43.00 38 113621652 26.1 22.5 22.4 29.0 0.0 36.1 24.5 smartseq 537038 SRR2088639 SRP060416 SRS979972 SRX1082608 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811080: T86_P3_H3_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811080 GSM1811080 T86_P3_H3_ILC1 103337471 2403197 2016-01-28 01:00:06 112481484 103337471 2403197 1 2403197 index:0,count:2403197,average:43,stdev:0 GSM1811080_r1 7.11 6.32 0.2 70549766 94632939 53193687 73860387 134.14 138.85 0 0 0 0 0 0 59.68 80.15 2915451 1021446 2915451 1021446 70.85 76.39 2915451 1212648 2915451 973502 7856889 11.14 6.14 0 18.19 0 1.12 0 0.28 0 0.00 0 27.38 0 1711670 0 43 0 41.74 0 1.14 0 0.01 0 1.14 0 0.01 0 52.75 0 0.34 0 147510 0 2403197 0 437206 0 26948 0 6661 0 0 0 657918 0 3 0 0 0 334 0 42769 0 786 0 43892 0 53.03 0 1274464 0 9628 55233 5.736705442459 2403197.0 1711670.0 147510.0 437206.0 26948.0 6661.0 0.0 657918.0 1274464.0 71.2 6.1 18.2 1.1 0.3 0.0 27.4 53.0 43 43 43.00 38 103337471 26.2 22.4 22.4 29.0 0.0 36.1 24.6 smartseq 537094 SRR2088640 SRP060416 SRS979971 SRX1082609 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811081: T86_P3_H4_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811081 GSM1811081 T86_P3_H4_ILC1 95259964 2215348 2016-01-28 01:00:06 103634551 95259964 2215348 1 2215348 index:0,count:2215348,average:43,stdev:0 GSM1811081_r1 6.5 6.58 0.25 59552762 77439663 45798419 61902961 130.04 135.16 0 0 0 0 0 0 57.93 76.48 2460036 840238 2460036 840238 66.5 72.79 2460036 964482 2460036 799655 8131152 13.65 7.41 0 15.88 0 1.06 0 0.37 0 0.00 0 33.10 0 1450429 0 43 0 41.69 0 1.18 0 0.01 0 1.10 0 0.01 0 295.38 0 0.35 0 164205 0 2215348 0 351814 0 23474 0 8114 0 0 0 733331 0 37 0 0 0 284 0 34954 0 807 0 36082 0 49.59 0 1098615 0 7974 43494 5.454477050414 2215348.0 1450429.0 164205.0 351814.0 23474.0 8114.0 0.0 733331.0 1098615.0 65.5 7.4 15.9 1.1 0.4 0.0 33.1 49.6 43 43 43.00 38 95259964 26.0 22.5 22.5 29.0 0.0 36.1 24.4 smartseq 537102 SRR2088641 SRP060416 SRS979970 SRX1082610 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811082: T86_P3_H5_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811082 GSM1811082 T86_P3_H5_ILC1 147374889 3427323 2016-01-28 01:00:06 159033208 147374889 3427323 1 3427323 index:0,count:3427323,average:43,stdev:0 GSM1811082_r1 8.04 6.64 0.22 88725448 114850643 68436564 92562359 129.44 135.25 0 0 0 0 0 0 56.42 74.41 3722393 1223447 3722393 1223447 64.22 71.61 3722393 1392731 3722393 1177319 14033694 15.82 7.93 0 15.30 0 0.99 0 0.34 0 0.00 0 35.40 0 2168641 0 43 0 41.62 0 1.15 0 0.01 0 1.13 0 0.01 0 224.33 0 0.35 0 271935 0 3427323 0 524502 0 33911 0 11569 0 0 0 1213202 0 17 0 0 0 397 0 43926 0 1127 0 45467 0 47.97 0 1644139 0 7223 53307 7.380174442752 3427323.0 2168641.0 271935.0 524502.0 33911.0 11569.0 0.0 1213202.0 1644139.0 63.3 7.9 15.3 1.0 0.3 0.0 35.4 48.0 43 43 43.00 38 147374889 26.2 22.4 22.4 29.0 0.0 36.1 24.4 smartseq 537110 SRR2088642 SRP060416 SRS979969 SRX1082611 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811083: T86_P3_H8_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811083 GSM1811083 T86_P3_H8_ILC1 70824268 1647076 2016-01-28 01:00:06 77702301 70824268 1647076 1 1647076 index:0,count:1647076,average:43,stdev:0 GSM1811083_r1 8.02 6.04 0.19 46562949 61672931 36662136 49880514 132.45 136.05 0 0 0 0 0 0 59.45 76.43 1812133 670695 1812133 670695 68.49 73.06 1812133 772682 1812133 641135 5987842 12.86 6.75 0 15.21 0 0.92 0 0.39 0 0.00 0 30.19 0 1128156 0 43 0 41.78 0 1.16 0 0.01 0 1.12 0 0.00 0 169.41 0 0.35 0 111116 0 1647076 0 250575 0 15130 0 6496 0 0 0 497294 0 5 0 0 0 236 0 27663 0 568 0 28472 0 53.28 0 877581 0 8479 33720 3.976884066517 1647076.0 1128156.0 111116.0 250575.0 15130.0 6496.0 0.0 497294.0 877581.0 68.5 6.7 15.2 0.9 0.4 0.0 30.2 53.3 43 43 43.00 38 70824268 26.0 22.8 22.9 28.3 0.0 36.0 24.5 smartseq 537118 SRR2088643 SRP060416 SRS979968 SRX1082612 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811084: T86_P3_H9_ILC1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC1|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811084 GSM1811084 T86_P3_H9_ILC1 41364710 961970 2016-01-28 01:00:06 45173640 41364710 961970 1 961970 index:0,count:961970,average:43,stdev:0 GSM1811084_r1 8.34 6.51 0.18 27658055 37763451 21930442 30882549 136.54 140.82 0 0 0 0 0 0 64.93 82.78 1051278 433364 1051278 433364 73.56 79.2 1051278 490998 1051278 414589 2732262 9.88 6.58 0 14.97 0 0.82 0 0.27 0 0.00 0 29.53 0 667468 0 43 0 41.89 0 1.11 0 0.01 0 1.09 0 0.00 0 182.27 0 0.33 0 63291 0 961970 0 143968 0 7868 0 2599 0 0 0 284035 0 6 0 0 0 138 0 18723 0 300 0 19167 0 54.42 0 523500 0 6933 21981 3.170488965816 961970.0 667468.0 63291.0 143968.0 7868.0 2599.0 0.0 284035.0 523500.0 69.4 6.6 15.0 0.8 0.3 0.0 29.5 54.4 43 43 43.00 38 41364710 25.8 23.1 23.2 27.9 0.0 36.2 25.0 smartseq 537126 SRR2088644 SRP060416 SRS979967 SRX1082613 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811085: T86_P4_A10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811085 GSM1811085 T86_P4_A10_ILC3 18131638 421666 2016-01-28 01:00:06 20656678 18131638 421666 1 421666 index:0,count:421666,average:43,stdev:0 GSM1811085_r1 6.25 7.06 0.18 12969202 16669064 10532849 13908083 128.53 132.04 0 0 0 0 0 0 58.67 73.32 477965 185149 477965 185149 64.22 69.46 477965 202669 477965 175403 2035967 15.70 5.33 0 14.95 0 0.98 0 0.42 0 0.00 0 23.76 0 315580 0 43 0 41.71 0 1.24 0 0.01 0 1.14 0 0.00 0 94.87 0 0.41 0 22461 0 421666 0 63044 0 4136 0 1770 0 0 0 100180 0 5 0 0 0 62 0 8136 0 151 0 8354 0 59.89 0 252536 0 4592 8848 1.926829268293 421666.0 315580.0 22461.0 63044.0 4136.0 1770.0 0.0 100180.0 252536.0 74.8 5.3 15.0 1.0 0.4 0.0 23.8 59.9 43 43 43.00 38 18131638 27.0 21.4 22.1 29.5 0.0 35.1 23.3 smartseq 537134 SRR2088645 SRP060416 SRS979966 SRX1082614 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811086: T86_P4_A11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811086 GSM1811086 T86_P4_A11_ILC3 52081944 1211208 2016-01-28 01:00:06 57804367 52081944 1211208 1 1211208 index:0,count:1211208,average:43,stdev:0 GSM1811086_r1 8.66 5.83 0.16 40279394 54061145 33056852 45426538 134.22 137.42 0 0 0 0 0 0 62.14 76.5 1425278 604824 1425278 604824 67.95 72.12 1425278 661404 1425278 570200 5118466 12.71 4.09 0 15.09 0 0.90 0 0.52 0 0.00 0 18.21 0 973334 0 43 0 41.81 0 1.20 0 0.01 0 1.13 0 0.00 0 335.41 0 0.37 0 49523 0 1211208 0 182734 0 10960 0 6317 0 0 0 220597 0 9 0 0 0 173 0 27225 0 443 0 27850 0 65.27 0 790600 0 9724 30784 3.165775401070 1211208.0 973334.0 49523.0 182734.0 10960.0 6317.0 0.0 220597.0 790600.0 80.4 4.1 15.1 0.9 0.5 0.0 18.2 65.3 43 43 43.00 38 52081944 26.9 22.0 22.2 28.9 0.0 35.8 24.3 smartseq 537142 SRR2088646 SRP060416 SRS979889 SRX1082615 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811087: T86_P4_A12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811087 GSM1811087 T86_P4_A12_ILC3 74613041 1735187 2016-01-28 01:00:06 84571797 74613041 1735187 1 1735187 index:0,count:1735187,average:43,stdev:0 GSM1811087_r1 7.17 7.16 0.2 54353381 69636714 43492654 57333099 128.12 131.82 0 0 0 0 0 0 58.72 74.42 2044539 774696 2044539 774696 65.33 70.89 2044539 861917 2044539 737915 7786843 14.33 5.13 0 16.04 0 0.99 0 0.51 0 0.00 0 22.46 0 1319354 0 43 0 41.78 0 1.21 0 0.01 0 1.16 0 0.01 0 231.36 0 0.42 0 89009 0 1735187 0 278363 0 17188 0 8884 0 0 0 389761 0 23 0 0 0 265 0 34410 0 604 0 35302 0 59.99 0 1040991 0 10869 39928 3.673567025485 1735187.0 1319354.0 89009.0 278363.0 17188.0 8884.0 0.0 389761.0 1040991.0 76.0 5.1 16.0 1.0 0.5 0.0 22.5 60.0 43 43 43.00 38 74613041 26.9 21.4 22.3 29.4 0.0 35.0 23.2 smartseq 537150 SRR2088647 SRP060416 SRS979965 SRX1082616 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811088: T86_P4_A1_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811088 GSM1811088 T86_P4_A1_ILC3 86885757 2020599 2016-01-28 01:00:06 97771857 86885757 2020599 1 2020599 index:0,count:2020599,average:43,stdev:0 GSM1811088_r1 5.77 7.22 0.22 62042317 79338980 50747661 66886000 127.88 131.8 0 0 0 0 0 0 57.45 71.35 2266690 867543 2266690 867543 62.14 67.71 2266690 938407 2266690 823290 9645795 15.55 5.35 0 14.56 0 1.12 0 0.48 0 0.00 0 23.67 0 1510109 0 43 0 41.74 0 1.21 0 0.01 0 1.18 0 0.01 0 213.95 0 0.42 0 108122 0 2020599 0 294214 0 22557 0 9690 0 0 0 478243 0 18 0 0 0 284 0 33798 0 608 0 34708 0 60.17 0 1215895 0 7649 38197 4.993724669891 2020599.0 1510109.0 108122.0 294214.0 22557.0 9690.0 0.0 478243.0 1215895.0 74.7 5.4 14.6 1.1 0.5 0.0 23.7 60.2 43 43 43.00 38 86885757 26.9 21.5 22.2 29.3 0.0 35.2 23.3 smartseq 537158 SRR2088648 SRP060416 SRS979964 SRX1082617 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811089: T86_P4_A3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811089 GSM1811089 T86_P4_A3_ILC3 120726026 2807582 2016-01-28 01:00:06 131545723 120726026 2807582 1 2807582 index:0,count:2807582,average:43,stdev:0 GSM1811089_r1 4.96 7.63 0.24 82956382 104769132 66864182 86842243 126.29 129.88 0 0 0 0 0 0 56.76 71.57 3120659 1147905 3120659 1147905 62.07 67.73 3120659 1255275 3120659 1086250 13684207 16.50 5.95 0 14.90 0 1.08 0 0.45 0 0.00 0 26.45 0 2022238 0 43 0 41.69 0 1.22 0 0.01 0 1.12 0 0.01 0 69.71 0 0.36 0 167164 0 2807582 0 418394 0 30223 0 12531 0 0 0 742590 0 9 0 0 0 370 0 49755 0 1013 0 51147 0 57.13 0 1603844 0 9013 57528 6.382780428270 2807582.0 2022238.0 167164.0 418394.0 30223.0 12531.0 0.0 742590.0 1603844.0 72.0 6.0 14.9 1.1 0.4 0.0 26.4 57.1 43 43 43.00 38 120726026 26.7 21.8 21.9 29.6 0.0 35.9 24.2 smartseq 537166 SRR2088649 SRP060416 SRS979890 SRX1082618 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811090: T86_P4_A5_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811090 GSM1811090 T86_P4_A5_ILC3 110608427 2572289 2016-01-28 01:00:06 121887947 110608427 2572289 1 2572289 index:0,count:2572289,average:43,stdev:0 GSM1811090_r1 4.79 7.33 0.21 76585652 98585931 62264266 82634997 128.73 132.72 0 0 0 0 0 0 58.17 72.67 2824369 1084404 2824369 1084404 63.36 69.08 2824369 1181055 2824369 1030863 11432439 14.93 5.81 0 14.45 0 1.13 0 0.51 0 0.00 0 25.89 0 1864063 0 43 0 41.72 0 1.20 0 0.01 0 1.12 0 0.01 0 280.61 0 0.37 0 149533 0 2572289 0 371790 0 29092 0 13183 0 0 0 665951 0 26 0 0 0 398 0 43837 0 970 0 45231 0 58.01 0 1492273 0 8959 51935 5.796963946869 2572289.0 1864063.0 149533.0 371790.0 29092.0 13183.0 0.0 665951.0 1492273.0 72.5 5.8 14.5 1.1 0.5 0.0 25.9 58.0 43 43 43.00 38 110608427 26.7 21.9 22.1 29.4 0.0 35.7 24.0 smartseq 537223 SRR2088650 SRP060416 SRS979891 SRX1082619 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811091: T86_P4_A8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811091 GSM1811091 T86_P4_A8_ILC3 109895487 2555709 2016-01-28 01:00:06 121124460 109895487 2555709 1 2555709 index:0,count:2555709,average:43,stdev:0 GSM1811091_r1 7.51 5.55 0.17 85564697 113394672 70716286 95786580 132.53 135.45 0 0 0 0 0 0 60.77 74.24 2968012 1250885 2968012 1250885 66.74 70.3 2968012 1373828 2968012 1184557 10955463 12.80 4.01 0 14.61 0 0.96 0 0.57 0 0.00 0 17.93 0 2058370 0 43 0 41.97 0 1.21 0 0.01 0 1.12 0 0.01 0 287.52 0 0.37 0 102506 0 2555709 0 373438 0 24636 0 14456 0 0 0 458247 0 28 0 0 0 383 0 52747 0 903 0 54061 0 65.93 0 1684932 0 11471 61725 5.380960683463 2555709.0 2058370.0 102506.0 373438.0 24636.0 14456.0 0.0 458247.0 1684932.0 80.5 4.0 14.6 1.0 0.6 0.0 17.9 65.9 43 43 43.00 38 109895487 26.8 22.2 22.5 28.5 0.0 35.8 24.4 smartseq 537231 SRR2088651 SRP060416 SRS979962 SRX1082620 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811092: T86_P4_A9_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811092 GSM1811092 T86_P4_A9_ILC3 27393150 637050 2016-01-28 01:00:06 30050798 27393150 637050 1 637050 index:0,count:637050,average:43,stdev:0 GSM1811092_r1 7.38 7.31 0.21 17147659 22014894 13735935 18186266 128.38 132.4 0 0 0 0 0 0 58.9 74.7 646732 245519 646732 245519 64.47 70.5 646732 268738 646732 231721 2583717 15.07 7.53 0 13.83 0 0.89 0 0.44 0 0.00 0 33.24 0 416818 0 43 0 41.79 0 1.17 0 0.01 0 1.12 0 0.00 0 120.70 0 0.35 0 47986 0 637050 0 88133 0 5670 0 2803 0 0 0 211759 0 8 0 0 0 93 0 11529 0 236 0 11866 0 51.59 0 328685 0 5714 12713 2.224886244312 637050.0 416818.0 47986.0 88133.0 5670.0 2803.0 0.0 211759.0 328685.0 65.4 7.5 13.8 0.9 0.4 0.0 33.2 51.6 43 43 43.00 38 27393150 26.7 21.9 22.0 29.4 0.0 36.1 24.5 smartseq 537287 SRR2088658 SRP060416 SRS979892 SRX1082627 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811099: T86_P4_B6_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811099 GSM1811099 T86_P4_B6_ILC3 172095933 4002231 2016-01-28 01:00:06 187823118 172095933 4002231 1 4002231 index:0,count:4002231,average:43,stdev:0 GSM1811099_r1 7.96 7.22 0.2 118476628 154826361 94608817 127198704 130.68 134.45 0 0 0 0 0 0 61.12 77.71 4457352 1763425 4457352 1763425 66.93 73.24 4457352 1931164 4457352 1661942 16449173 13.88 5.96 0 15.39 0 0.95 0 0.38 0 0.00 0 26.58 0 2885393 0 43 0 41.69 0 1.19 0 0.01 0 1.13 0 0.00 0 327.46 0 0.36 0 238731 0 4002231 0 616136 0 38011 0 15069 0 0 0 1063758 0 28 0 0 0 656 0 81321 0 1352 0 83357 0 56.70 0 2269257 0 12536 94171 7.512045309509 4002231.0 2885393.0 238731.0 616136.0 38011.0 15069.0 0.0 1063758.0 2269257.0 72.1 6.0 15.4 0.9 0.4 0.0 26.6 56.7 43 43 43.00 38 172095933 26.5 21.9 22.1 29.5 0.0 35.8 24.1 smartseq 537791 SRR2088697 SRP060416 SRS979923 SRX1082666 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811138: T86_P4_F12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811138 GSM1811138 T86_P4_F12_ILC3 117463659 2731713 2016-01-28 01:00:06 127458069 117463659 2731713 1 2731713 index:0,count:2731713,average:43,stdev:0 GSM1811138_r1 7.06 6.16 0.21 86134431 113104142 70843117 95356280 131.31 134.6 0 0 0 0 0 0 61.11 75.12 3079725 1273333 3079725 1273333 67.39 71.6 3079725 1404195 3079725 1213585 11023739 12.80 4.98 0 14.23 0 1.05 0 0.51 0 0.00 0 22.17 0 2083627 0 43 0 41.79 0 1.18 0 0.01 0 1.11 0 0.00 0 298.01 0 0.34 0 135990 0 2731713 0 388604 0 28561 0 13922 0 0 0 605603 0 31 0 0 0 411 0 56071 0 1019 0 57532 0 62.05 0 1695023 0 11462 65163 5.685133484558 2731713.0 2083627.0 135990.0 388604.0 28561.0 13922.0 0.0 605603.0 1695023.0 76.3 5.0 14.2 1.0 0.5 0.0 22.2 62.0 43 43 43.00 38 117463659 26.5 22.4 22.4 28.8 0.0 36.1 24.7 smartseq 538631 SRR2088700 SRP060416 SRS979921 SRX1082669 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811141: T86_P4_F3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811141 GSM1811141 T86_P4_F3_ILC3 164319641 3821387 2016-01-28 01:00:06 177401312 164319641 3821387 1 3821387 index:0,count:3821387,average:43,stdev:0 GSM1811141_r1 8.34 5.55 0.22 123743653 162739395 101707541 136252773 131.51 133.97 0 0 0 0 0 0 62.44 76.76 4369963 1863699 4369963 1863699 68.71 72.77 4369963 2050714 4369963 1766901 15402397 12.45 4.58 0 14.56 0 0.95 0 0.52 0 0.00 0 20.43 0 2984560 0 43 0 41.89 0 1.19 0 0.01 0 1.15 0 0.00 0 305.71 0 0.33 0 175041 0 3821387 0 556540 0 36243 0 19974 0 0 0 780610 0 44 0 0 0 681 0 85688 0 1287 0 87700 0 63.54 0 2428020 0 13908 100026 7.191975841242 3821387.0 2984560.0 175041.0 556540.0 36243.0 19974.0 0.0 780610.0 2428020.0 78.1 4.6 14.6 0.9 0.5 0.0 20.4 63.5 43 43 43.00 38 164319641 26.6 22.3 22.3 28.8 0.0 36.1 24.9 smartseq 538639 SRR2088701 SRP060416 SRS979919 SRX1082670 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811142: T86_P4_F4_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811142 GSM1811142 T86_P4_F4_ILC3 110439523 2568361 2016-01-28 01:00:06 120112745 110439523 2568361 1 2568361 index:0,count:2568361,average:43,stdev:0 GSM1811142_r1 6.3 6.31 0.21 80884405 106544751 66669334 89929470 131.72 134.89 0 0 0 0 0 0 63.85 78.45 2859273 1251894 2859273 1251894 68.75 74.05 2859273 1347916 2859273 1181669 9943600 12.29 5.00 0 14.20 0 0.94 0 0.50 0 0.00 0 22.22 0 1960590 0 43 0 41.78 0 1.18 0 0.01 0 1.16 0 0.00 0 308.20 0 0.34 0 128383 0 2568361 0 364763 0 24213 0 12776 0 0 0 570782 0 3 0 0 0 440 0 54701 0 837 0 55981 0 62.13 0 1595827 0 9774 62574 6.402087170043 2568361.0 1960590.0 128383.0 364763.0 24213.0 12776.0 0.0 570782.0 1595827.0 76.3 5.0 14.2 0.9 0.5 0.0 22.2 62.1 43 43 43.00 38 110439523 26.6 22.2 22.2 29.0 0.0 36.1 24.5 smartseq 538783 SRR2088713 SRP060416 SRS979910 SRX1082682 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811154: T86_P4_G8_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811154 GSM1811154 T86_P4_G8_ILC3 106583283 2478681 2016-01-28 01:00:06 115855185 106583283 2478681 1 2478681 index:0,count:2478681,average:43,stdev:0 GSM1811154_r1 4.59 6.5 0.17 76125756 96907352 61925559 80900662 127.3 130.64 0 0 0 0 0 0 59.61 74.21 2779018 1099652 2779018 1099652 65.66 70.43 2779018 1211084 2779018 1043516 10720843 14.08 5.31 0 14.64 0 1.22 0 0.51 0 0.00 0 23.85 0 1844618 0 43 0 41.79 0 1.20 0 0.01 0 1.14 0 0.00 0 262.45 0 0.35 0 131638 0 2478681 0 362880 0 30357 0 12535 0 0 0 591171 0 36 0 0 0 301 0 49722 0 911 0 50970 0 59.78 0 1481738 0 9721 59009 6.070260261290 2478681.0 1844618.0 131638.0 362880.0 30357.0 12535.0 0.0 591171.0 1481738.0 74.4 5.3 14.6 1.2 0.5 0.0 23.9 59.8 43 43 43.00 38 106583283 26.5 22.3 22.4 28.8 0.0 36.1 24.5 smartseq 538791 SRR2088714 SRP060416 SRS979909 SRX1082683 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811155: T86_P4_G9_NK; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;NK|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811155 GSM1811155 T86_P4_G9_NK 35206422 818754 2016-01-28 01:00:06 38474933 35206422 818754 1 818754 index:0,count:818754,average:43,stdev:0 GSM1811155_r1 7.05 5.83 0.19 24725620 32847082 20249520 27561615 132.85 136.11 0 0 0 0 0 0 61.25 75.6 893707 365396 893707 365396 67.82 72.15 893707 404627 893707 348733 3162894 12.79 5.84 0 13.83 0 1.01 0 0.45 0 0.00 0 25.68 0 596598 0 43 0 41.89 0 1.19 0 0.01 0 1.15 0 0.01 0 27.81 0 0.34 0 47817 0 818754 0 113257 0 8278 0 3652 0 0 0 210226 0 14 0 0 0 123 0 16243 0 245 0 16625 0 59.03 0 483341 0 6579 18469 2.807265541876 818754.0 596598.0 47817.0 113257.0 8278.0 3652.0 0.0 210226.0 483341.0 72.9 5.8 13.8 1.0 0.4 0.0 25.7 59.0 43 43 43.00 38 35206422 26.7 22.3 22.4 28.7 0.0 36.2 24.9 smartseq 538799 SRR2088715 SRP060416 SRS979908 SRX1082684 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811156: T86_P4_H10_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811156 GSM1811156 T86_P4_H10_ILC3 43029713 1000691 2016-01-28 01:00:06 48884323 43029713 1000691 1 1000691 index:0,count:1000691,average:43,stdev:0 GSM1811156_r1 6.46 9.01 0.22 26879810 33297468 20429776 26323837 123.88 128.85 0 0 0 0 0 0 54.0 72.6 1112335 357887 1112335 357887 60.58 68.74 1112335 401477 1112335 338856 4547544 16.92 7.41 0 16.97 0 1.17 0 0.40 0 0.00 0 32.20 0 662739 0 43 0 41.45 0 1.23 0 0.01 0 1.13 0 0.00 0 211.91 0 0.43 0 74103 0 1000691 0 169807 0 11693 0 4050 0 0 0 322209 0 15 0 0 0 128 0 15490 0 313 0 15946 0 49.26 0 492932 0 6121 17926 2.928606436857 1000691.0 662739.0 74103.0 169807.0 11693.0 4050.0 0.0 322209.0 492932.0 66.2 7.4 17.0 1.2 0.4 0.0 32.2 49.3 43 43 43.00 38 43029713 26.5 21.0 21.8 30.6 0.0 34.9 22.9 smartseq 538807 SRR2088716 SRP060416 SRS979906 SRX1082685 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811157: T86_P4_H11_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811157 GSM1811157 T86_P4_H11_ILC3 117858270 2740890 2016-01-28 01:00:06 128458671 117858270 2740890 1 2740890 index:0,count:2740890,average:43,stdev:0 GSM1811157_r1 4.94 7.62 0.23 79217762 99488996 63934592 82469403 125.59 128.99 0 0 0 0 0 0 58.46 73.64 2966181 1130812 2966181 1130812 64.03 70.24 2966181 1238505 2966181 1078596 12138811 15.32 6.29 0 14.54 0 1.06 0 0.42 0 0.00 0 27.96 0 1934198 0 43 0 41.63 0 1.19 0 0.01 0 1.14 0 0.00 0 259.66 0 0.36 0 172496 0 2740890 0 398512 0 28940 0 11505 0 0 0 766247 0 12 0 0 0 464 0 48468 0 1040 0 49984 0 56.03 0 1535686 0 8026 55516 6.917019686020 2740890.0 1934198.0 172496.0 398512.0 28940.0 11505.0 0.0 766247.0 1535686.0 70.6 6.3 14.5 1.1 0.4 0.0 28.0 56.0 43 43 43.00 38 117858270 26.7 21.8 21.9 29.5 0.0 35.9 24.2 smartseq 538815 SRR2088717 SRP060416 SRS979907 SRX1082686 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811158: T86_P4_H12_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811158 GSM1811158 T86_P4_H12_ILC3 135109784 3142088 2016-01-28 01:00:06 146822388 135109784 3142088 1 3142088 index:0,count:3142088,average:43,stdev:0 GSM1811158_r1 4.79 7.57 0.23 89258152 113198608 71418893 94106694 126.82 131.77 0 0 0 0 0 0 58.2 73.94 3420366 1267333 3420366 1267333 63.63 70.66 3420366 1385632 3420366 1211021 13157584 14.74 6.41 0 14.75 0 1.68 0 0.51 0 0.00 0 28.50 0 2177543 0 43 0 41.67 0 1.23 0 0.01 0 1.11 0 0.01 0 269.32 0 0.35 0 201514 0 3142088 0 463580 0 52879 0 16026 0 0 0 895640 0 16 0 0 0 364 0 47221 0 1210 0 48811 0 54.55 0 1713963 0 6895 55596 8.063234227701 3142088.0 2177543.0 201514.0 463580.0 52879.0 16026.0 0.0 895640.0 1713963.0 69.3 6.4 14.8 1.7 0.5 0.0 28.5 54.5 43 43 43.00 38 135109784 26.7 21.9 22.0 29.4 0.0 36.0 24.2 smartseq 538823 SRR2088718 SRP060416 SRS979904 SRX1082687 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811159: T86_P4_H2_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811159 GSM1811159 T86_P4_H2_ILC3 33038577 768339 2016-01-28 01:00:06 37584739 33038577 768339 1 768339 index:0,count:768339,average:43,stdev:0 GSM1811159_r1 5.04 7.48 0.2 23264312 29419291 18439161 23983542 126.46 130.07 0 0 0 0 0 0 60.01 76.99 893042 341682 893042 341682 66.52 73.31 893042 378765 893042 325317 3189063 13.71 5.50 0 16.35 0 1.07 0 0.39 0 0.00 0 24.43 0 569397 0 43 0 41.55 0 1.21 0 0.01 0 1.20 0 0.00 0 162.71 0 0.43 0 42295 0 768339 0 125617 0 8211 0 3012 0 0 0 187719 0 4 0 0 0 108 0 14918 0 272 0 15302 0 57.76 0 443780 0 5733 17516 3.055293912437 768339.0 569397.0 42295.0 125617.0 8211.0 3012.0 0.0 187719.0 443780.0 74.1 5.5 16.3 1.1 0.4 0.0 24.4 57.8 43 43 43.00 38 33038577 27.1 21.2 21.9 29.8 0.0 35.0 23.0 smartseq 538831 SRR2088719 SRP060416 SRS979903 SRX1082688 SRA275902 GEO Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs) from three independent tonsil donors. Overall design: Sequencing libraries were prepared from FACS sorted individual ILCs with the Smart-Seq2 protocol (Picelli et al. Nature Methods 2013) GSM1811160: T86_P4_H3_ILC3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single cDNA was generated from FACS sorted cells using to Smart-seq2 (Picelli et al. Nature Methods 2013) Nextera XT DNA Sample Preparation Index Kit (24 index primers, Illumina) Illumina HiSeq 2000 cell type;;tonsil Innate lymphoid cells|facs gating;;ILC3|source_name;;cDNA|tonsil donor;;Donor T86 GEO Accession;;GSM1811160 GSM1811160 T86_P4_H3_ILC3 122647266 2852262 2016-01-28 01:00:06 133753366 122647266 2852262 1 2852262 index:0,count:2852262,average:43,stdev:0 GSM1811160_r1 4.66 9.53 0.27 69897243 85428000 52263389 66783692 122.22 127.78 0 0 0 0 0 0 52.92 72.61 3006568 920720 3006568 920720 59.47 69.15 3006568 1034633 3006568 876877 13103247 18.75 8.48 0 16.54 0 1.26 0 0.31 0 0.00 0 37.44 0 1739734 0 43 0 41.22 0 1.20 0 0.01 0 1.20 0 0.00 0 270.21 0 0.38 0 241736 0 2852262 0 471691 0 35850 0 8894 0 0 0 1067784 0 17 0 0 0 291 0 37529 0 1129 0 38966 0 44.46 0 1268043 0 6263 43666 6.972058119112 2852262.0 1739734.0 241736.0 471691.0 35850.0 8894.0 0.0 1067784.0 1268043.0 61.0 8.5 16.5 1.3 0.3 0.0 37.4 44.5 43 43 43.00 38 122647266 26.3 21.3 21.4 31.1 0.0 35.7 23.6 smartseq 1145098 SRR2149790 SRP062177 SRS1028575 SRX1137097 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846954: Live_PLRC [US-1457350-15]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846954 GSM1846954 Live_PLRC [US-1457350-15] 593611338 5819719 2015-10-05 11:31:16 379173721 593611338 5819719 2 5819719 index:0,count:5819719,average:51,stdev:0|index:1,count:5819719,average:51,stdev:0 GSM1846954_r1 4.41 3.66 0.19 515993940 701745145 474621266 647371390 136.0 136.4 5457610 4259421 228.981 2046.597 80 20360 83.57 91.13 6168635 4560741 6168635 4560741 85.82 86.38 6168635 4683755 6168635 4323054 20101082 3.90 1.15 0 7.78 0 0.08 0 0.07 0 0.00 0 6.07 0 5457610 0 102 0 100.26 0 1.43 0 0.01 0 1.22 0 0.01 0 272.09 0 0.26 0 66827 0 5819719 0 452745 0 4836 0 3805 0 0 0 353468 0 638 0 0 0 5106 0 890618 0 4358 0 900720 0 86.00 0 5004865 0 58400 918476 15.727328767123 5819719.0 5457610.0 66827.0 452745.0 4836.0 3805.0 0.0 353468.0 5004865.0 93.8 1.1 7.8 0.1 0.1 0.0 6.1 86.0 51 51 51.00 38 296805669 26.4 23.1 23.3 27.3 0.0 38.0 25.4 smartseq 1145129 SRR2149792 SRP062177 SRS1028573 SRX1137099 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846956: Live_PLRC [US-1457350-17]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846956 GSM1846956 Live_PLRC [US-1457350-17] 668282478 6551789 2015-10-05 11:31:16 426495403 668282478 6551789 2 6551789 index:0,count:6551789,average:51,stdev:0|index:1,count:6551789,average:51,stdev:0 GSM1846956_r1 5.87 3.79 0.23 589163666 804719269 545516722 747150133 136.59 136.96 6209718 4802708 234.876 2260.806 90 22399 83.65 90.6 6950702 5194556 6950702 5194556 85.73 86.25 6950702 5323537 6950702 4944790 25084144 4.26 1.13 0 7.27 0 0.10 0 0.07 0 0.00 0 5.06 0 6209718 0 102 0 100.34 0 1.45 0 0.01 0 1.24 0 0.01 0 228.99 0 0.26 0 73920 0 6551789 0 476392 0 6396 0 4478 0 0 0 331197 0 672 0 0 0 6308 0 1001362 0 4758 0 1013100 0 87.51 0 5733326 0 69456 1032756 14.869212163096 6551789.0 6209718.0 73920.0 476392.0 6396.0 4478.0 0.0 331197.0 5733326.0 94.8 1.1 7.3 0.1 0.1 0.0 5.1 87.5 51 51 51.00 38 334141239 26.4 23.0 23.2 27.3 0.0 38.0 25.5 smartseq 1145192 SRR2149796 SRP062177 SRS1028569 SRX1137103 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846960: Fixed_PLRC [US-1457350-20]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846960 GSM1846960 Fixed_PLRC [US-1457350-20] 779653830 7643665 2015-10-05 11:31:16 490234672 779653830 7643665 2 7643665 index:0,count:7643665,average:51,stdev:0|index:1,count:7643665,average:51,stdev:0 GSM1846960_r1 5.43 3.66 0.26 687369842 939492689 630229738 864538921 136.68 137.18 7209836 5465411 248.307 2456.973 89 23551 82.26 89.97 8176420 5930908 8176420 5930908 84.75 85.32 8176420 6110623 8176420 5624662 27594324 4.01 1.10 0 8.08 0 0.10 0 0.09 0 0.00 0 5.48 0 7209836 0 102 0 100.25 0 1.76 0 0.02 0 1.21 0 0.01 0 252.45 0 0.25 0 84216 0 7643665 0 617744 0 7647 0 7170 0 0 0 419012 0 861 0 0 0 7411 0 1135709 0 8187 0 1152168 0 86.24 0 6592092 0 58639 1178378 20.095465475196 7643665.0 7209836.0 84216.0 617744.0 7647.0 7170.0 0.0 419012.0 6592092.0 94.3 1.1 8.1 0.1 0.1 0.0 5.5 86.2 51 51 51.00 38 389826915 26.4 23.0 23.2 27.4 0.0 38.1 25.4 smartseq 1145225 SRR2149798 SRP062177 SRS1028567 SRX1137105 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846962: Fixed_PLRC [US-1457350-22]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846962 GSM1846962 Fixed_PLRC [US-1457350-22] 610966434 5989867 2015-10-05 11:31:16 385594572 610966434 5989867 2 5989867 index:0,count:5989867,average:51,stdev:0|index:1,count:5989867,average:51,stdev:0 GSM1846962_r1 5.71 3.57 0.26 539750794 742833020 492175956 680866072 137.63 138.34 5657192 4322549 246.503 2310.777 90 18682 83.32 91.62 6461792 4713437 6461792 4713437 86.26 86.9 6461792 4879822 6461792 4470299 19230369 3.56 1.09 0 8.56 0 0.10 0 0.08 0 0.00 0 5.38 0 5657192 0 102 0 100.26 0 1.80 0 0.02 0 1.21 0 0.01 0 253.69 0 0.26 0 65518 0 5989867 0 512881 0 5944 0 4768 0 0 0 321963 0 636 0 0 0 6491 0 894828 0 7127 0 909082 0 85.88 0 5144311 0 57450 932030 16.223324630113 5989867.0 5657192.0 65518.0 512881.0 5944.0 4768.0 0.0 321963.0 5144311.0 94.4 1.1 8.6 0.1 0.1 0.0 5.4 85.9 51 51 51.00 38 305483217 26.2 23.2 23.4 27.2 0.0 38.1 25.4 smartseq 1145241 SRR2149799 SRP062177 SRS1028566 SRX1137106 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846963: Fixed_PLRC [US-1457350-23]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846963 GSM1846963 Fixed_PLRC [US-1457350-23] 639046116 6265158 2015-10-05 11:31:16 402806643 639046116 6265158 2 6265158 index:0,count:6265158,average:51,stdev:0|index:1,count:6265158,average:51,stdev:0 GSM1846963_r1 8.16 3.49 0.22 550362977 758344103 507344821 701444991 137.79 138.26 5762886 4513532 246.564 2162.729 91 19227 81.63 88.81 6477763 4704349 6477763 4704349 84.07 84.54 6477763 4845080 6477763 4478213 27098628 4.92 1.05 0 7.43 0 0.11 0 0.11 0 0.00 0 7.79 0 5762886 0 102 0 100.38 0 1.74 0 0.02 0 1.21 0 0.01 0 227.82 0 0.25 0 65974 0 6265158 0 465539 0 6969 0 7157 0 0 0 488146 0 572 0 0 0 5090 0 815524 0 6254 0 827440 0 84.55 0 5297347 0 43986 848472 19.289592142955 6265158.0 5762886.0 65974.0 465539.0 6969.0 7157.0 0.0 488146.0 5297347.0 92.0 1.1 7.4 0.1 0.1 0.0 7.8 84.6 51 51 51.00 38 319523058 26.9 22.5 22.7 27.9 0.0 38.1 25.4 smartseq 1147144 SRR2149810 SRP062177 SRS1028555 SRX1137118 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846974: Live_PLRC [US-1457351-12]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846974 GSM1846974 Live_PLRC [US-1457351-12] 737334132 7228766 2015-10-05 11:31:16 466739211 737334132 7228766 2 7228766 index:0,count:7228766,average:51,stdev:0|index:1,count:7228766,average:51,stdev:0 GSM1846974_r1 5.79 3.55 0.28 519660131 713128123 472618685 652366532 137.23 138.03 5520913 4362671 223.817 2058.170 79 22442 83.11 91.68 6347910 4588363 6347910 4588363 86.1 86.86 6347910 4753482 6347910 4347520 16468693 3.17 0.98 0 7.14 0 0.09 0 0.07 0 0.00 0 23.46 0 5520913 0 102 0 100.19 0 1.43 0 0.01 0 1.21 0 0.01 0 185.88 0 0.27 0 71009 0 7228766 0 515965 0 6817 0 5096 0 0 0 1695940 0 569 0 0 0 5724 0 876155 0 5193 0 887641 0 69.24 0 5004948 0 55511 907250 16.343607573274 7228766.0 5520913.0 71009.0 515965.0 6817.0 5096.0 0.0 1695940.0 5004948.0 76.4 1.0 7.1 0.1 0.1 0.0 23.5 69.2 51 51 51.00 38 368667066 25.6 23.8 24.0 26.6 0.0 37.9 24.5 smartseq 1147208 SRR2149814 SRP062177 SRS1028551 SRX1137122 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846978: Fixed_PLRC [US-1457351-16]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846978 GSM1846978 Fixed_PLRC [US-1457351-16] 743880696 7292948 2015-10-05 11:31:16 461241311 743880696 7292948 2 7292948 index:0,count:7292948,average:51,stdev:0|index:1,count:7292948,average:51,stdev:0 GSM1846978_r1 5.31 3.49 0.26 528411678 725818939 478254393 660968712 137.36 138.2 5686907 4597412 202.960 1818.656 78 27258 83.04 92.1 6604586 4722386 6604586 4722386 86.12 86.91 6604586 4897607 6604586 4456685 17047235 3.23 1.01 0 7.67 0 0.12 0 0.08 0 0.00 0 21.82 0 5686907 0 102 0 100.10 0 1.78 0 0.02 0 1.18 0 0.01 0 181.07 0 0.27 0 73689 0 7292948 0 559252 0 8910 0 5583 0 0 0 1591548 0 536 0 0 0 5385 0 917499 0 7650 0 931070 0 70.31 0 5127655 0 65502 944140 14.413911025618 7292948.0 5686907.0 73689.0 559252.0 8910.0 5583.0 0.0 1591548.0 5127655.0 78.0 1.0 7.7 0.1 0.1 0.0 21.8 70.3 51 51 51.00 38 371940348 26.2 23.3 23.3 27.1 0.0 38.1 25.6 smartseq 1147224 SRR2149815 SRP062177 SRS1028663 SRX1137123 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846979: Fixed_PLRC [US-1457351-17]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846979 GSM1846979 Fixed_PLRC [US-1457351-17] 775995702 7607801 2015-10-05 11:31:16 487452106 775995702 7607801 2 7607801 index:0,count:7607801,average:51,stdev:0|index:1,count:7607801,average:51,stdev:0 GSM1846979_r1 6.95 3.3 0.27 537820438 730810080 491033105 669360480 135.88 136.32 5721613 4635938 218.962 1883.410 78 24032 79.28 87.1 6557680 4536030 6557680 4536030 82.25 82.52 6557680 4705838 6557680 4297176 30316144 5.64 1.02 0 6.76 0 0.10 0 0.09 0 0.00 0 24.60 0 5721613 0 102 0 100.17 0 1.75 0 0.02 0 1.19 0 0.01 0 165.99 0 0.27 0 77532 0 7607801 0 513921 0 7643 0 6807 0 0 0 1871738 0 568 0 0 0 5213 0 829153 0 7552 0 842486 0 68.45 0 5207692 0 53336 861023 16.143374081296 7607801.0 5721613.0 77532.0 513921.0 7643.0 6807.0 0.0 1871738.0 5207692.0 75.2 1.0 6.8 0.1 0.1 0.0 24.6 68.5 51 51 51.00 38 387997851 25.8 23.6 23.7 26.9 0.0 38.0 24.8 smartseq 1147241 SRR2149816 SRP062177 SRS1028662 SRX1137124 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846980: Fixed_PLRC [US-1457351-18]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846980 GSM1846980 Fixed_PLRC [US-1457351-18] 613639344 6016072 2015-10-05 11:31:16 382541400 613639344 6016072 2 6016072 index:0,count:6016072,average:51,stdev:0|index:1,count:6016072,average:51,stdev:0 GSM1846980_r1 5.68 3.54 0.26 483416920 664707510 439624781 606717825 137.5 138.01 5143962 4140495 218.990 1932.301 78 21710 81.66 90.08 5916656 4200627 5916656 4200627 84.47 84.88 5916656 4345291 5916656 3958335 20884695 4.32 1.12 0 7.99 0 0.10 0 0.08 0 0.00 0 14.32 0 5143962 0 102 0 100.10 0 1.85 0 0.03 0 1.20 0 0.01 0 171.89 0 0.27 0 67665 0 6016072 0 480759 0 5811 0 4822 0 0 0 861477 0 498 0 0 0 4921 0 776914 0 8219 0 790552 0 77.51 0 4663203 0 47485 802733 16.904980520164 6016072.0 5143962.0 67665.0 480759.0 5811.0 4822.0 0.0 861477.0 4663203.0 85.5 1.1 8.0 0.1 0.1 0.0 14.3 77.5 51 51 51.00 38 306819672 25.9 23.5 23.5 27.1 0.0 38.0 24.9 smartseq 1147257 SRR2149817 SRP062177 SRS1028661 SRX1137125 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846981: Fixed_PLRC [US-1457351-19]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846981 GSM1846981 Fixed_PLRC [US-1457351-19] 781727796 7663998 2015-10-05 11:31:16 495422511 781727796 7663998 2 7663998 index:0,count:7663998,average:51,stdev:0|index:1,count:7663998,average:51,stdev:0 GSM1846981_r1 5.74 3.58 0.29 655167444 898023646 595952029 820677125 137.07 137.71 6994907 5596860 212.648 1879.789 78 30892 82.54 91.03 8038638 5773358 8038638 5773358 85.64 86.23 8038638 5990206 8038638 5468550 24248161 3.70 1.16 0 8.52 0 0.09 0 0.08 0 0.00 0 8.55 0 6994907 0 102 0 100.15 0 1.78 0 0.02 0 1.19 0 0.01 0 250.82 0 0.28 0 89222 0 7663998 0 652777 0 7203 0 6457 0 0 0 655431 0 922 0 0 0 6366 0 1102015 0 9462 0 1118765 0 82.75 0 6342130 0 56965 1140896 20.028017203546 7663998.0 6994907.0 89222.0 652777.0 7203.0 6457.0 0.0 655431.0 6342130.0 91.3 1.2 8.5 0.1 0.1 0.0 8.6 82.8 51 51 51.00 38 390863898 26.1 23.3 23.5 27.1 0.0 38.0 25.3 smartseq 1147273 SRR2149818 SRP062177 SRS1028659 SRX1137126 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846982: Live_RLT [US-1457351-1]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846982 GSM1846982 Live_RLT [US-1457351-1] 803513772 7877586 2015-10-05 11:31:16 506741628 803513772 7877586 2 7877586 index:0,count:7877586,average:51,stdev:0|index:1,count:7877586,average:51,stdev:0 GSM1846982_r1 18.91 2.92 0.19 702937865 1020905944 647393331 948296955 145.23 146.48 7463480 6189910 218.921 1853.077 78 30278 82.54 90.0 8622484 6160502 8622484 6160502 85.16 86.13 8622484 6355766 8622484 5895882 33512139 4.77 1.15 0 7.85 0 0.10 0 0.11 0 0.00 0 5.04 0 7463480 0 102 0 100.37 0 1.25 0 0.01 0 1.19 0 0.01 0 329.76 0 0.26 0 90594 0 7877586 0 618488 0 8183 0 8573 0 0 0 397350 0 717 0 0 0 6254 0 954045 0 4688 0 965704 0 86.89 0 6844992 0 66337 981544 14.796327841175 7877586.0 7463480.0 90594.0 618488.0 8183.0 8573.0 0.0 397350.0 6844992.0 94.7 1.2 7.9 0.1 0.1 0.0 5.0 86.9 51 51 51.00 38 401756886 26.6 22.9 22.9 27.6 0.0 38.0 25.2 smartseq 1147291 SRR2149819 SRP062177 SRS1028660 SRX1137127 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846983: Fixed_RLT [US-1457351-20]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_Triton X Lysis|source_name;;H1 ECSs p40 Fixed Cells Triton X Lysis GEO Accession;;GSM1846983 GSM1846983 Fixed_RLT [US-1457351-20] 645605634 6329467 2015-10-05 11:31:16 404553987 645605634 6329467 2 6329467 index:0,count:6329467,average:51,stdev:0|index:1,count:6329467,average:51,stdev:0 GSM1846983_r1 21.16 1.7 0.13 542449076 795254936 472736723 726566735 146.6 153.69 5797666 5227317 210.320 1410.814 81 25906 77.33 89.2 8564360 4483050 8564360 4483050 80.51 85.16 8564360 4667803 8564360 4279950 30926558 5.70 1.43 0 12.20 0 0.14 0 0.07 0 0.00 0 8.19 0 5797666 0 102 0 99.92 0 1.41 0 0.02 0 1.16 0 0.01 0 237.36 0 0.30 0 90474 0 6329467 0 772034 0 9095 0 4199 0 0 0 518507 0 554 0 0 0 2179 0 419625 0 4729 0 427087 0 79.40 0 5025632 0 8588 438836 51.098742431299 6329467.0 5797666.0 90474.0 772034.0 9095.0 4199.0 0.0 518507.0 5025632.0 91.6 1.4 12.2 0.1 0.1 0.0 8.2 79.4 51 51 51.00 38 322802817 26.2 22.7 22.8 28.4 0.0 37.9 23.2 smartseq 1147403 SRR2149820 SRP062177 SRS1028658 SRX1137128 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846984: Fixed_RLT [US-1457351-21]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_Triton X Lysis|source_name;;H1 ECSs p40 Fixed Cells Triton X Lysis GEO Accession;;GSM1846984 GSM1846984 Fixed_RLT [US-1457351-21] 801420120 7857060 2015-10-05 11:31:16 504459186 801420120 7857060 2 7857060 index:0,count:7857060,average:51,stdev:0|index:1,count:7857060,average:51,stdev:0 GSM1846984_r1 14.76 1.49 0.1 605345853 859337689 499408212 759932440 141.96 152.17 7177315 6810281 126.086 963.327 69 71928 74.9 91.53 12299658 5376165 12299658 5376165 78.79 86.11 12299658 5654819 12299658 5057751 21175261 3.50 1.93 0 16.59 0 0.37 0 0.12 0 0.00 0 8.16 0 7177315 0 102 0 99.58 0 1.40 0 0.02 0 1.16 0 0.01 0 233.76 0 0.29 0 151862 0 7857060 0 1303841 0 28757 0 9497 0 0 0 641491 0 708 0 0 0 3124 0 612195 0 5342 0 621369 0 74.75 0 5873474 0 7715 603329 78.202073882048 7857060.0 7177315.0 151862.0 1303841.0 28757.0 9497.0 0.0 641491.0 5873474.0 91.3 1.9 16.6 0.4 0.1 0.0 8.2 74.8 51 51 51.00 38 400710060 24.7 24.8 24.4 26.1 0.0 37.8 24.9 smartseq 1147418 SRR2149821 SRP062177 SRS1028622 SRX1137129 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846985: Fixed_RLT [US-1457351-22]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_Triton X Lysis|source_name;;H1 ECSs p40 Fixed Cells Triton X Lysis GEO Accession;;GSM1846985 GSM1846985 Fixed_RLT [US-1457351-22] 771842058 7567079 2015-10-05 11:31:16 484777373 771842058 7567079 2 7567079 index:0,count:7567079,average:51,stdev:0|index:1,count:7567079,average:51,stdev:0 GSM1846985_r1 16.91 1.57 0.08 623274196 895654642 534765241 811390831 143.7 151.73 6824216 6295129 174.645 1112.685 81 40570 78.99 92.59 10622776 5390745 10622776 5390745 82.18 88.03 10622776 5608211 10622776 5125534 19252436 3.09 1.38 0 13.24 0 0.19 0 0.08 0 0.00 0 9.55 0 6824216 0 102 0 100.00 0 1.39 0 0.02 0 1.13 0 0.01 0 223.29 0 0.28 0 104141 0 7567079 0 1001834 0 14403 0 5765 0 0 0 722695 0 137 0 0 0 2083 0 495236 0 5609 0 503065 0 76.94 0 5822382 0 6606 504939 76.436421435059 7567079.0 6824216.0 104141.0 1001834.0 14403.0 5765.0 0.0 722695.0 5822382.0 90.2 1.4 13.2 0.2 0.1 0.0 9.6 76.9 51 51 51.00 38 385921029 25.8 23.3 23.2 27.6 0.0 37.9 23.8 smartseq 1147434 SRR2149822 SRP062177 SRS1028621 SRX1137130 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846986: Live_RLT [US-1457351-2]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846986 GSM1846986 Live_RLT [US-1457351-2] 800585658 7848879 2015-10-05 11:31:16 501579700 800585658 7848879 2 7848879 index:0,count:7848879,average:51,stdev:0|index:1,count:7848879,average:51,stdev:0 GSM1846986_r1 9.0 2.78 0.22 683100630 946534383 619057694 872550586 138.56 140.95 7242877 5777899 223.965 2024.044 78 29536 83.36 92.33 9038968 6037368 9038968 6037368 85.89 87.93 9038968 6221247 9038968 5750177 24577928 3.60 1.11 0 8.96 0 0.11 0 0.04 0 0.00 0 7.56 0 7242877 0 102 0 100.34 0 1.28 0 0.01 0 1.19 0 0.01 0 227.87 0 0.25 0 87028 0 7848879 0 703651 0 8817 0 3523 0 0 0 593662 0 727 0 0 0 7101 0 1108386 0 5726 0 1121940 0 83.31 0 6539226 0 70403 1143067 16.236055281735 7848879.0 7242877.0 87028.0 703651.0 8817.0 3523.0 0.0 593662.0 6539226.0 92.3 1.1 9.0 0.1 0.0 0.0 7.6 83.3 51 51 51.00 38 400292829 26.1 23.4 23.4 27.1 0.0 38.0 25.1 smartseq 1147452 SRR2149823 SRP062177 SRS1028620 SRX1137131 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846987: Live_RLT [US-1457351-3]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846987 GSM1846987 Live_RLT [US-1457351-3] 663278052 6502726 2015-10-05 11:31:16 422848260 663278052 6502726 2 6502726 index:0,count:6502726,average:51,stdev:0|index:1,count:6502726,average:51,stdev:0 GSM1846987_r1 11.32 2.96 0.2 590024426 825777941 540560667 766754020 139.96 141.84 6230085 4959244 221.762 1916.527 88 25297 84.77 92.85 7502103 5281212 7502103 5281212 86.95 88.67 7502103 5417126 7502103 5043352 20766380 3.52 1.01 0 8.33 0 0.08 0 0.04 0 0.00 0 4.07 0 6230085 0 102 0 100.41 0 1.27 0 0.01 0 1.19 0 0.01 0 236.46 0 0.27 0 65863 0 6502726 0 541992 0 4963 0 2773 0 0 0 264905 0 614 0 0 0 5742 0 957681 0 4412 0 968449 0 87.47 0 5688093 0 74489 987041 13.250828981460 6502726.0 6230085.0 65863.0 541992.0 4963.0 2773.0 0.0 264905.0 5688093.0 95.8 1.0 8.3 0.1 0.0 0.0 4.1 87.5 51 51 51.00 38 331639026 26.7 22.9 23.1 27.3 0.0 38.0 25.7 smartseq 1147466 SRR2149824 SRP062177 SRS1028619 SRX1137132 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846988: Live_RLT [US-1457351-4]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846988 GSM1846988 Live_RLT [US-1457351-4] 666211572 6531486 2015-10-05 11:31:16 417631041 666211572 6531486 2 6531486 index:0,count:6531486,average:51,stdev:0|index:1,count:6531486,average:51,stdev:0 GSM1846988_r1 10.05 3.07 0.21 590499846 817686434 538224163 757024266 138.47 140.65 6211020 4877703 241.440 2111.382 78 22613 81.91 90.18 7606264 5087445 7606264 5087445 84.42 86.18 7606264 5243206 7606264 4861791 29189037 4.94 1.09 0 8.72 0 0.12 0 0.08 0 0.00 0 4.70 0 6211020 0 102 0 100.42 0 1.29 0 0.01 0 1.19 0 0.01 0 237.51 0 0.25 0 71112 0 6531486 0 569802 0 7805 0 5436 0 0 0 307225 0 716 0 0 0 5431 0 917149 0 4735 0 928031 0 86.37 0 5641218 0 68177 948057 13.905818677853 6531486.0 6211020.0 71112.0 569802.0 7805.0 5436.0 0.0 307225.0 5641218.0 95.1 1.1 8.7 0.1 0.1 0.0 4.7 86.4 51 51 51.00 38 333105786 26.5 23.0 23.1 27.4 0.0 38.1 25.5 smartseq 1147483 SRR2149825 SRP062177 SRS1028618 SRX1137133 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846989: Live_RLT [US-1457351-5]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846989 GSM1846989 Live_RLT [US-1457351-5] 632727012 6203206 2015-10-05 11:31:16 395819708 632727012 6203206 2 6203206 index:0,count:6203206,average:51,stdev:0|index:1,count:6203206,average:51,stdev:0 GSM1846989_r1 9.06 2.79 0.22 554074191 764417643 500569581 703778362 137.96 140.6 5858799 4635202 232.215 2118.144 78 22573 82.56 91.74 7396173 4837016 7396173 4837016 85.16 87.31 7396173 4989092 7396173 4603464 23186223 4.18 1.17 0 9.45 0 0.11 0 0.06 0 0.00 0 5.38 0 5858799 0 102 0 100.29 0 1.28 0 0.01 0 1.20 0 0.01 0 250.92 0 0.25 0 72463 0 6203206 0 586368 0 6737 0 3970 0 0 0 333700 0 705 0 0 0 5427 0 877062 0 4190 0 887384 0 85.00 0 5272431 0 74664 905974 12.134013714776 6203206.0 5858799.0 72463.0 586368.0 6737.0 3970.0 0.0 333700.0 5272431.0 94.4 1.2 9.5 0.1 0.1 0.0 5.4 85.0 51 51 51.00 38 316363506 26.3 23.2 23.2 27.3 0.0 38.0 25.1 smartseq 1147497 SRR2149826 SRP062177 SRS1028617 SRX1137134 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846990: Live_RLT [US-1457351-6]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846990 GSM1846990 Live_RLT [US-1457351-6] 805868034 7900667 2015-10-05 11:31:16 502456508 805868034 7900667 2 7900667 index:0,count:7900667,average:51,stdev:0|index:1,count:7900667,average:51,stdev:0 GSM1846990_r1 8.09 2.94 0.2 698703766 961032000 632340959 885176671 137.54 139.98 7443851 5970260 218.129 1923.799 78 31144 81.8 90.74 9316247 6088811 9316247 6088811 84.38 86.33 9316247 6280821 9316247 5792893 31473632 4.50 1.18 0 9.29 0 0.13 0 0.07 0 0.00 0 5.58 0 7443851 0 102 0 100.26 0 1.28 0 0.01 0 1.18 0 0.01 0 273.48 0 0.25 0 93144 0 7900667 0 733883 0 10318 0 5627 0 0 0 440871 0 835 0 0 0 6785 0 1153649 0 6123 0 1167392 0 84.93 0 6709968 0 76328 1184970 15.524709149984 7900667.0 7443851.0 93144.0 733883.0 10318.0 5627.0 0.0 440871.0 6709968.0 94.2 1.2 9.3 0.1 0.1 0.0 5.6 84.9 51 51 51.00 38 402934017 26.0 23.5 23.5 27.0 0.0 38.1 25.5 smartseq 1147512 SRR2149827 SRP062177 SRS1028616 SRX1137135 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846991: Live_PLRC [US-1457351-7]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846991 GSM1846991 Live_PLRC [US-1457351-7] 775313424 7601112 2015-10-05 11:31:16 496179515 775313424 7601112 2 7601112 index:0,count:7601112,average:51,stdev:0|index:1,count:7601112,average:51,stdev:0 GSM1846991_r1 4.59 3.31 0.26 533342348 736482862 490015138 678980239 138.09 138.56 5657183 4448392 220.763 1979.657 88 23852 86.26 94.16 6400039 4879979 6400039 4879979 88.65 89.25 6400039 5015012 6400039 4625503 11806604 2.21 0.88 0 6.24 0 0.06 0 0.03 0 0.00 0 25.48 0 5657183 0 102 0 100.28 0 1.44 0 0.01 0 1.20 0 0.01 0 165.84 0 0.27 0 66596 0 7601112 0 474439 0 4366 0 2606 0 0 0 1936957 0 746 0 0 0 5904 0 953721 0 5336 0 965707 0 68.18 0 5182744 0 60521 983019 16.242610003139 7601112.0 5657183.0 66596.0 474439.0 4366.0 2606.0 0.0 1936957.0 5182744.0 74.4 0.9 6.2 0.1 0.0 0.0 25.5 68.2 51 51 51.00 38 387656712 25.5 24.0 24.1 26.3 0.0 37.9 24.9 smartseq 1147528 SRR2149828 SRP062177 SRS1028615 SRX1137136 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846992: Live_PLRC [US-1457351-8]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846992 GSM1846992 Live_PLRC [US-1457351-8] 682586142 6692021 2015-10-05 11:31:16 429617286 682586142 6692021 2 6692021 index:0,count:6692021,average:51,stdev:0|index:1,count:6692021,average:51,stdev:0 GSM1846992_r1 4.52 3.54 0.23 553981356 765999038 503433540 699317057 138.27 138.91 5926210 4738679 210.430 1839.030 78 27018 84.76 93.58 6823968 5023226 6823968 5023226 87.76 88.46 6823968 5201077 6823968 4748405 13561085 2.45 1.12 0 8.35 0 0.10 0 0.06 0 0.00 0 11.28 0 5926210 0 102 0 100.22 0 1.45 0 0.01 0 1.19 0 0.01 0 213.20 0 0.26 0 74912 0 6692021 0 558573 0 6472 0 4270 0 0 0 755069 0 632 0 0 0 6404 0 1000774 0 5691 0 1013501 0 80.21 0 5367637 0 65830 1030917 15.660291660337 6692021.0 5926210.0 74912.0 558573.0 6472.0 4270.0 0.0 755069.0 5367637.0 88.6 1.1 8.3 0.1 0.1 0.0 11.3 80.2 51 51 51.00 38 341293071 25.7 23.8 23.8 26.7 0.0 38.0 24.9 smartseq 1147544 SRR2149829 SRP062177 SRS1028614 SRX1137137 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846993: Live_PLRC [US-1457351-9]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846993 GSM1846993 Live_PLRC [US-1457351-9] 777404832 7621616 2015-10-05 11:31:16 493240193 777404832 7621616 2 7621616 index:0,count:7621616,average:51,stdev:0|index:1,count:7621616,average:51,stdev:0 GSM1846993_r1 5.57 3.61 0.23 500343637 696000446 455138086 635742868 139.1 139.68 5334706 4212614 211.872 1796.979 80 24247 85.36 94.11 6124777 4553523 6124777 4553523 88.3 88.91 6124777 4710623 6124777 4301951 11476431 2.29 0.81 0 6.51 0 0.07 0 0.06 0 0.00 0 29.87 0 5334706 0 102 0 100.25 0 1.46 0 0.01 0 1.17 0 0.01 0 125.29 0 0.26 0 61494 0 7621616 0 496288 0 5691 0 4345 0 0 0 2276874 0 668 0 0 0 5581 0 947554 0 5063 0 958866 0 63.48 0 4838418 0 68124 980074 14.386618519171 7621616.0 5334706.0 61494.0 496288.0 5691.0 4345.0 0.0 2276874.0 4838418.0 70.0 0.8 6.5 0.1 0.1 0.0 29.9 63.5 51 51 51.00 38 388702416 25.1 24.7 24.8 25.4 0.0 38.0 25.9 smartseq 572462 SRR2149785 SRP062177 SRS1028580 SRX1137092 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846949: Live_PLRC [US-1457350-10]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846949 GSM1846949 Live_PLRC [US-1457350-10] 720595932 7064666 2015-10-05 11:31:16 451623052 720595932 7064666 2 7064666 index:0,count:7064666,average:51,stdev:0|index:1,count:7064666,average:51,stdev:0 GSM1846949_r1 2.54 3.72 0.24 626594317 837711670 576331261 773597977 133.69 134.23 6618846 5183819 232.185 2163.950 90 24636 82.58 90.04 7520785 5466072 7520785 5466072 85.02 85.68 7520785 5627029 7520785 5201384 27054034 4.32 1.12 0 7.75 0 0.10 0 0.06 0 0.00 0 6.16 0 6618846 0 102 0 100.34 0 1.46 0 0.01 0 1.23 0 0.01 0 249.34 0 0.24 0 78922 0 7064666 0 547863 0 6792 0 4152 0 0 0 434876 0 720 0 0 0 6836 0 1040999 0 5326 0 1053881 0 85.93 0 6070983 0 57878 1078445 18.633073015654 7064666.0 6618846.0 78922.0 547863.0 6792.0 4152.0 0.0 434876.0 6070983.0 93.7 1.1 7.8 0.1 0.1 0.0 6.2 85.9 51 51 51.00 38 360297966 26.8 22.7 22.8 27.8 0.0 38.1 25.4 smartseq 572470 SRR2149786 SRP062177 SRS1028579 SRX1137093 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846950: Live_PLRC [US-1457350-11]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846950 GSM1846950 Live_PLRC [US-1457350-11] 502676604 4928202 2015-10-05 11:31:16 312729533 502676604 4928202 2 4928202 index:0,count:4928202,average:51,stdev:0|index:1,count:4928202,average:51,stdev:0 GSM1846950_r1 3.97 3.7 0.27 438166775 598873181 402150824 551756741 136.68 137.2 4675408 3756831 208.425 1785.749 78 21707 84.03 91.85 5305613 3928666 5305613 3928666 86.2 86.86 5305613 4030215 5305613 3715244 15246616 3.48 1.15 0 8.08 0 0.09 0 0.07 0 0.00 0 4.98 0 4675408 0 102 0 100.31 0 1.44 0 0.01 0 1.22 0 0.01 0 272.95 0 0.24 0 56496 0 4928202 0 398075 0 4204 0 3337 0 0 0 245253 0 509 0 0 0 4610 0 784691 0 3769 0 793579 0 86.79 0 4277333 0 56973 805058 14.130517964650 4928202.0 4675408.0 56496.0 398075.0 4204.0 3337.0 0.0 245253.0 4277333.0 94.9 1.1 8.1 0.1 0.1 0.0 5.0 86.8 51 51 51.00 38 251338302 26.1 23.4 23.4 27.1 0.0 38.1 25.4 smartseq 572478 SRR2149787 SRP062177 SRS1028578 SRX1137094 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846951: Live_PLRC [US-1457350-12]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846951 GSM1846951 Live_PLRC [US-1457350-12] 777659424 7624112 2015-10-05 11:31:16 483537859 777659424 7624112 2 7624112 index:0,count:7624112,average:51,stdev:0|index:1,count:7624112,average:51,stdev:0 GSM1846951_r1 6.72 3.6 0.23 668458557 915250383 618580511 849400054 136.92 137.31 7084985 5564902 226.380 2099.442 78 27154 82.72 89.67 7931396 5860995 7931396 5860995 84.72 85.27 7931396 6002711 7931396 5572946 30067283 4.50 1.16 0 7.20 0 0.08 0 0.07 0 0.00 0 6.92 0 7084985 0 102 0 100.30 0 1.46 0 0.01 0 1.21 0 0.01 0 256.51 0 0.23 0 88220 0 7624112 0 549103 0 6165 0 5616 0 0 0 527346 0 977 0 0 0 7157 0 1112613 0 4884 0 1125631 0 85.73 0 6535882 0 52902 1146701 21.675947979282 7624112.0 7084985.0 88220.0 549103.0 6165.0 5616.0 0.0 527346.0 6535882.0 92.9 1.2 7.2 0.1 0.1 0.0 6.9 85.7 51 51 51.00 38 388829712 26.5 23.0 23.0 27.6 0.0 38.1 25.4 smartseq 572486 SRR2149788 SRP062177 SRS1028577 SRX1137095 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846952: Live_PLRC [US-1457350-13]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846952 GSM1846952 Live_PLRC [US-1457350-13] 775402368 7601984 2015-10-05 11:31:16 484375447 775402368 7601984 2 7601984 index:0,count:7601984,average:51,stdev:0|index:1,count:7601984,average:51,stdev:0 GSM1846952_r1 2.78 3.82 0.23 680533471 919036883 626710432 849826027 135.05 135.6 7159512 5501204 246.354 2369.628 79 25015 82.57 89.92 8104626 5911861 8104626 5911861 84.95 85.5 8104626 6081660 8104626 5621224 27807436 4.09 1.14 0 7.69 0 0.10 0 0.08 0 0.00 0 5.64 0 7159512 0 102 0 100.33 0 1.45 0 0.01 0 1.22 0 0.01 0 255.77 0 0.25 0 86619 0 7601984 0 584892 0 7881 0 5890 0 0 0 428701 0 744 0 0 0 7627 0 1116766 0 6051 0 1131188 0 86.49 0 6574620 0 67503 1155533 17.118246596448 7601984.0 7159512.0 86619.0 584892.0 7881.0 5890.0 0.0 428701.0 6574620.0 94.2 1.1 7.7 0.1 0.1 0.0 5.6 86.5 51 51 51.00 38 387701184 26.6 22.9 23.0 27.6 0.0 38.1 25.4 smartseq 572495 SRR2149789 SRP062177 SRS1028576 SRX1137096 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846953: Live_PLRC [US-1457350-14]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846953 GSM1846953 Live_PLRC [US-1457350-14] 645513426 6328563 2015-10-05 11:31:16 414021965 645513426 6328563 2 6328563 index:0,count:6328563,average:51,stdev:0|index:1,count:6328563,average:51,stdev:0 GSM1846953_r1 3.38 3.72 0.21 553630197 747735969 508323966 689205870 135.06 135.58 5822291 4449063 241.000 2301.068 78 20336 83.2 90.87 6604615 4844196 6604615 4844196 85.62 86.27 6604615 4985097 6604615 4599418 20521839 3.71 1.07 0 7.76 0 0.10 0 0.09 0 0.00 0 7.81 0 5822291 0 102 0 100.32 0 1.46 0 0.01 0 1.21 0 0.01 0 214.93 0 0.28 0 67784 0 6328563 0 491147 0 6043 0 5661 0 0 0 494568 0 637 0 0 0 6194 0 953893 0 4591 0 965315 0 84.24 0 5331144 0 66370 989586 14.910140123550 6328563.0 5822291.0 67784.0 491147.0 6043.0 5661.0 0.0 494568.0 5331144.0 92.0 1.1 7.8 0.1 0.1 0.0 7.8 84.2 51 51 51.00 38 322756713 26.4 23.0 23.2 27.4 0.0 38.0 25.2 smartseq 572559 SRR2149791 SRP062177 SRS1028574 SRX1137098 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846955: Live_PLRC [US-1457350-16]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846955 GSM1846955 Live_PLRC [US-1457350-16] 654755136 6419168 2015-10-05 11:31:16 423796215 654755136 6419168 2 6419168 index:0,count:6419168,average:51,stdev:0|index:1,count:6419168,average:51,stdev:0 GSM1846955_r1 5.75 3.57 0.36 522119529 709088576 478354002 652143784 135.81 136.33 5497522 4216958 242.009 2302.847 79 19236 82.35 90.15 6257046 4526956 6257046 4526956 85.15 85.72 6257046 4681284 6257046 4304644 21287625 4.08 1.06 0 7.41 0 0.11 0 0.09 0 0.00 0 14.16 0 5497522 0 102 0 100.25 0 1.44 0 0.01 0 1.22 0 0.01 0 204.50 0 0.28 0 67768 0 6419168 0 475954 0 6855 0 5907 0 0 0 908884 0 605 0 0 0 5984 0 892717 0 5074 0 904380 0 78.23 0 5021568 0 76885 925696 12.040007803863 6419168.0 5497522.0 67768.0 475954.0 6855.0 5907.0 0.0 908884.0 5021568.0 85.6 1.1 7.4 0.1 0.1 0.0 14.2 78.2 51 51 51.00 38 327377568 26.0 23.4 23.5 27.1 0.0 38.0 25.0 smartseq 572575 SRR2149793 SRP062177 SRS1028572 SRX1137100 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846957: Fixed_PLRC [US-1457350-18]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846957 GSM1846957 Fixed_PLRC [US-1457350-18] 502265442 4924171 2015-10-05 11:31:16 318892492 502265442 4924171 2 4924171 index:0,count:4924171,average:51,stdev:0|index:1,count:4924171,average:51,stdev:0 GSM1846957_r1 6.05 3.58 0.26 434166425 598876660 395386487 547709725 137.94 138.53 4605882 3612242 223.655 2035.357 78 17898 83.95 92.49 5276556 3866613 5276556 3866613 86.79 87.54 5276556 3997224 5276556 3659746 13991039 3.22 1.16 0 8.63 0 0.09 0 0.07 0 0.00 0 6.31 0 4605882 0 102 0 100.16 0 1.82 0 0.02 0 1.21 0 0.01 0 253.24 0 0.26 0 57278 0 4924171 0 425139 0 4190 0 3277 0 0 0 310822 0 519 0 0 0 4588 0 726380 0 5560 0 737047 0 84.90 0 4180743 0 51407 752011 14.628571984360 4924171.0 4605882.0 57278.0 425139.0 4190.0 3277.0 0.0 310822.0 4180743.0 93.5 1.2 8.6 0.1 0.1 0.0 6.3 84.9 51 51 51.00 38 251132721 26.7 22.7 22.9 27.7 0.0 38.1 25.4 smartseq 572583 SRR2149794 SRP062177 SRS1028571 SRX1137101 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846958: Fixed_PLRC [US-1457350-19]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846958 GSM1846958 Fixed_PLRC [US-1457350-19] 663676362 6506631 2015-10-05 11:31:16 423069377 663676362 6506631 2 6506631 index:0,count:6506631,average:51,stdev:0|index:1,count:6506631,average:51,stdev:0 GSM1846958_r1 3.04 3.8 0.23 567562167 776620761 518501117 713346253 136.83 137.58 6103393 4902377 210.122 1950.562 80 27415 81.24 89.24 7014637 4958198 7014637 4958198 83.37 84.05 7014637 5088314 7014637 4669768 24996644 4.40 1.29 0 8.41 0 0.16 0 0.12 0 0.00 0 5.91 0 6103393 0 102 0 100.08 0 1.81 0 0.02 0 1.23 0 0.01 0 218.91 0 0.28 0 84019 0 6506631 0 547128 0 10374 0 8118 0 0 0 384746 0 557 0 0 0 5425 0 933811 0 7427 0 947220 0 85.39 0 5556265 0 39759 959989 24.145199828970 6506631.0 6103393.0 84019.0 547128.0 10374.0 8118.0 0.0 384746.0 5556265.0 93.8 1.3 8.4 0.2 0.1 0.0 5.9 85.4 51 51 51.00 38 331838181 26.1 23.3 23.4 27.1 0.0 38.0 25.5 smartseq 572591 SRR2149795 SRP062177 SRS1028570 SRX1137102 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846959: Live_RLT [US-1457350-1]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846959 GSM1846959 Live_RLT [US-1457350-1] 923665182 9055541 2015-10-05 11:31:16 580430593 923665182 9055541 2 9055541 index:0,count:9055541,average:51,stdev:0|index:1,count:9055541,average:51,stdev:0 GSM1846959_r1 6.3 3.37 0.2 790390363 1076351019 723259569 995960642 136.18 137.7 8657725 7299439 179.119 1501.573 79 52644 80.13 87.93 10431631 6937257 10431631 6937257 81.53 82.83 10431631 7059006 10431631 6535031 47584059 6.02 1.27 0 8.48 0 0.14 0 0.11 0 0.00 0 4.14 0 8657725 0 102 0 100.31 0 1.33 0 0.01 0 1.20 0 0.00 0 313.46 0 0.24 0 115030 0 9055541 0 767987 0 12435 0 10304 0 0 0 375077 0 989 0 0 0 9107 0 1452099 0 5602 0 1467797 0 87.13 0 7889738 0 74290 1476208 19.870884372055 9055541.0 8657725.0 115030.0 767987.0 12435.0 10304.0 0.0 375077.0 7889738.0 95.6 1.3 8.5 0.1 0.1 0.0 4.1 87.1 51 51 51.00 38 461832591 25.1 24.6 24.5 25.8 0.0 38.0 26.4 smartseq 572607 SRR2149797 SRP062177 SRS1028568 SRX1137104 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846961: Fixed_PLRC [US-1457350-21]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846961 GSM1846961 Fixed_PLRC [US-1457350-21] 652766544 6399672 2015-10-05 11:31:16 409380238 652766544 6399672 2 6399672 index:0,count:6399672,average:51,stdev:0|index:1,count:6399672,average:51,stdev:0 GSM1846961_r1 4.06 3.82 0.24 571166980 781019246 522154090 717139265 136.74 137.34 5998410 4608115 245.494 2333.881 78 20222 82.26 90.23 6846906 4934397 6846906 4934397 84.79 85.4 6846906 5085851 6846906 4670458 23242171 4.07 1.16 0 8.27 0 0.12 0 0.08 0 0.00 0 6.07 0 5998410 0 102 0 100.19 0 1.84 0 0.02 0 1.23 0 0.01 0 253.17 0 0.26 0 74372 0 6399672 0 529460 0 7515 0 5136 0 0 0 388611 0 666 0 0 0 5816 0 913039 0 7430 0 926951 0 85.46 0 5468950 0 56332 952516 16.908968259604 6399672.0 5998410.0 74372.0 529460.0 7515.0 5136.0 0.0 388611.0 5468950.0 93.7 1.2 8.3 0.1 0.1 0.0 6.1 85.5 51 51 51.00 38 326383272 26.6 22.8 23.0 27.6 0.0 38.1 25.3 smartseq 573446 SRR2149800 SRP062177 SRS1028565 SRX1137107 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846964: Live_RLT [US-1457350-2]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846964 GSM1846964 Live_RLT [US-1457350-2] 809291052 7934226 2015-10-05 11:31:16 510902142 809291052 7934226 2 7934226 index:0,count:7934226,average:51,stdev:0|index:1,count:7934226,average:51,stdev:0 GSM1846964_r1 6.28 3.13 0.19 696890911 934901131 640179116 867938551 134.15 135.58 7575959 6338264 183.737 1620.962 78 42872 77.5 84.69 9052085 5871540 9052085 5871540 78.83 79.96 9052085 5972185 9052085 5543357 54048884 7.76 1.20 0 8.11 0 0.16 0 0.16 0 0.00 0 4.20 0 7575959 0 102 0 100.36 0 1.34 0 0.01 0 1.21 0 0.00 0 294.47 0 0.24 0 95564 0 7934226 0 643075 0 12299 0 12818 0 0 0 333150 0 867 0 0 0 7810 0 1191230 0 4482 0 1204389 0 87.38 0 6932884 0 58250 1211888 20.804944206009 7934226.0 7575959.0 95564.0 643075.0 12299.0 12818.0 0.0 333150.0 6932884.0 95.5 1.2 8.1 0.2 0.2 0.0 4.2 87.4 51 51 51.00 38 404645526 25.8 23.8 23.8 26.6 0.0 38.1 26.2 smartseq 573455 SRR2149801 SRP062177 SRS1028564 SRX1137108 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846965: Live_RLT [US-1457350-3]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846965 GSM1846965 Live_RLT [US-1457350-3] 680764626 6674163 2015-10-05 11:31:16 426088099 680764626 6674163 2 6674163 index:0,count:6674163,average:51,stdev:0|index:1,count:6674163,average:51,stdev:0 GSM1846965_r1 5.45 3.21 0.16 600208586 801414506 553746251 746554951 133.52 134.82 6400960 5152226 207.249 1975.798 78 29710 78.69 85.57 7563943 5037205 7563943 5037205 80.06 81.18 7563943 5124612 7563943 4778877 45413169 7.57 1.05 0 7.71 0 0.13 0 0.12 0 0.00 0 3.85 0 6400960 0 102 0 100.45 0 1.38 0 0.01 0 1.21 0 0.01 0 304.14 0 0.23 0 70330 0 6674163 0 514386 0 8514 0 7890 0 0 0 256799 0 695 0 0 0 6638 0 1018889 0 4127 0 1030349 0 88.20 0 5886574 0 67632 1044680 15.446534185001 6674163.0 6400960.0 70330.0 514386.0 8514.0 7890.0 0.0 256799.0 5886574.0 95.9 1.1 7.7 0.1 0.1 0.0 3.8 88.2 51 51 51.00 38 340382313 26.0 23.5 23.6 26.9 0.0 38.1 25.8 smartseq 573463 SRR2149802 SRP062177 SRS1028563 SRX1137109 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846966: Live_RLT [US-1457350-4]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846966 GSM1846966 Live_RLT [US-1457350-4] 712137684 6981742 2015-10-05 11:31:16 445909210 712137684 6981742 2 6981742 index:0,count:6981742,average:51,stdev:0|index:1,count:6981742,average:51,stdev:0 GSM1846966_r1 3.96 3.44 0.21 594865142 777028362 553987166 728072356 130.62 131.42 6309055 5140327 216.037 1941.677 81 27337 73.89 79.57 7246778 4661954 7246778 4661954 75.01 75.54 7246778 4732440 7246778 4425569 66884130 11.24 0.99 0 6.45 0 0.11 0 0.11 0 0.00 0 9.41 0 6309055 0 102 0 100.54 0 1.40 0 0.01 0 1.24 0 0.01 0 228.49 0 0.23 0 68999 0 6981742 0 450432 0 7590 0 7777 0 0 0 657320 0 555 0 0 0 6351 0 894075 0 4115 0 905096 0 83.91 0 5858623 0 54050 916892 16.963774283071 6981742.0 6309055.0 68999.0 450432.0 7590.0 7777.0 0.0 657320.0 5858623.0 90.4 1.0 6.5 0.1 0.1 0.0 9.4 83.9 51 51 51.00 38 356068842 26.4 23.1 23.1 27.4 0.0 38.1 25.3 smartseq 573470 SRR2149803 SRP062177 SRS1028562 SRX1137110 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846967: Live_RLT [US-1457350-5]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_Triton X Lysis|source_name;;H1 ESCs p40 Live Cells Triton X Lysis GEO Accession;;GSM1846967 GSM1846967 Live_RLT [US-1457350-5] 747964368 7332984 2015-10-05 11:31:16 469428274 747964368 7332984 2 7332984 index:0,count:7332984,average:51,stdev:0|index:1,count:7332984,average:51,stdev:0 GSM1846967_r1 5.46 3.57 0.2 662688387 878489589 612873553 818737207 132.56 133.59 6999369 5604906 223.356 2110.331 90 28732 75.96 82.37 8160155 5316483 8160155 5316483 77.45 78.26 8160155 5421318 8160155 5050731 61436090 9.27 1.04 0 7.44 0 0.13 0 0.14 0 0.00 0 4.29 0 6999369 0 102 0 100.48 0 1.36 0 0.01 0 1.22 0 0.01 0 296.62 0 0.24 0 76268 0 7332984 0 545300 0 9267 0 10110 0 0 0 314238 0 803 0 0 0 6744 0 1026555 0 4287 0 1038389 0 88.01 0 6454069 0 60451 1058194 17.504987510546 7332984.0 6999369.0 76268.0 545300.0 9267.0 10110.0 0.0 314238.0 6454069.0 95.5 1.0 7.4 0.1 0.1 0.0 4.3 88.0 51 51 51.00 38 373982184 26.5 23.0 23.1 27.5 0.0 38.1 25.3 smartseq 573479 SRR2149804 SRP062177 SRS1028561 SRX1137111 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846968: Fixed_RLT [US-1457350-6]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_Triton X Lysis|source_name;;H1 ECSs p40 Fixed Cells Triton X Lysis GEO Accession;;GSM1846968 GSM1846968 Fixed_RLT [US-1457350-6] 830970438 8146769 2015-10-05 11:31:16 519393446 830970438 8146769 2 8146769 index:0,count:8146769,average:51,stdev:0|index:1,count:8146769,average:51,stdev:0 GSM1846968_r1 19.55 1.16 0.15 668353965 952424419 574410887 866170914 142.5 150.79 7469642 7095076 155.123 864.356 69 53437 78.0 91.31 11802880 5826015 11802880 5826015 80.79 86.88 11802880 6034639 11802880 5543211 25087361 3.75 1.63 0 13.37 0 0.17 0 0.06 0 0.00 0 8.08 0 7469642 0 102 0 99.93 0 1.39 0 0.02 0 1.14 0 0.01 0 225.60 0 0.26 0 132520 0 8146769 0 1089177 0 13979 0 5280 0 0 0 657868 0 387 0 0 0 2047 0 403897 0 5359 0 411690 0 78.32 0 6380465 0 4270 409267 95.847072599532 8146769.0 7469642.0 132520.0 1089177.0 13979.0 5280.0 0.0 657868.0 6380465.0 91.7 1.6 13.4 0.2 0.1 0.0 8.1 78.3 51 51 51.00 38 415485219 26.3 22.7 22.6 28.3 0.0 37.8 23.4 smartseq 573487 SRR2149805 SRP062177 SRS1028560 SRX1137112 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846969: Fixed_RLT [US-1457350-7]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_Triton X Lysis|source_name;;H1 ECSs p40 Fixed Cells Triton X Lysis GEO Accession;;GSM1846969 GSM1846969 Fixed_RLT [US-1457350-7] 837233952 8208176 2015-10-05 11:31:16 522100848 837233952 8208176 2 8208176 index:0,count:8208176,average:51,stdev:0|index:1,count:8208176,average:51,stdev:0 GSM1846969_r1 11.37 1.81 0.08 702956525 958776487 595783231 863196773 136.39 144.88 7577860 6841056 191.492 1306.560 100 39696 76.51 90.69 12336247 5798127 12336247 5798127 79.86 86.38 12336247 6051582 12336247 5522241 29785828 4.24 1.34 0 14.43 0 0.15 0 0.06 0 0.00 0 7.47 0 7577860 0 102 0 99.92 0 1.48 0 0.03 0 1.15 0 0.01 0 250.42 0 0.28 0 110270 0 8208176 0 1184732 0 12581 0 4959 0 0 0 612776 0 260 0 0 0 2911 0 642457 0 6761 0 652389 0 77.89 0 6393128 0 7512 663063 88.267172523962 8208176.0 7577860.0 110270.0 1184732.0 12581.0 4959.0 0.0 612776.0 6393128.0 92.3 1.3 14.4 0.2 0.1 0.0 7.5 77.9 51 51 51.00 38 418616976 25.9 23.1 23.2 27.8 0.0 37.8 23.4 smartseq 573494 SRR2149806 SRP062177 SRS1028559 SRX1137113 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846970: Fixed_RLT [US-1457350-8]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_Triton X Lysis|source_name;;H1 ECSs p40 Fixed Cells Triton X Lysis GEO Accession;;GSM1846970 GSM1846970 Fixed_RLT [US-1457350-8] 863200092 8462746 2015-10-05 11:31:16 552660151 863200092 8462746 2 8462746 index:0,count:8462746,average:51,stdev:0|index:1,count:8462746,average:51,stdev:0 GSM1846970_r1 19.92 1.17 0.09 465713877 683529155 377780423 611353313 146.77 161.83 4982497 4539273 214.207 1313.820 78 22830 72.57 90.1 9302223 3615933 9302223 3615933 77.36 86.73 9302223 3854406 9302223 3480444 21375640 4.59 0.85 0 11.45 0 0.13 0 0.04 0 0.00 0 40.95 0 4982497 0 102 0 99.95 0 1.39 0 0.03 0 1.15 0 0.01 0 125.37 0 0.31 0 72315 0 8462746 0 969312 0 11033 0 3593 0 0 0 3465623 0 339 0 0 0 2152 0 329302 0 5654 0 337447 0 47.42 0 4013185 0 18477 345589 18.703739784597 8462746.0 4982497.0 72315.0 969312.0 11033.0 3593.0 0.0 3465623.0 4013185.0 58.9 0.9 11.5 0.1 0.0 0.0 41.0 47.4 51 51 51.00 38 431600046 25.4 23.8 24.0 26.9 0.0 37.7 22.8 smartseq 573502 SRR2149807 SRP062177 SRS1028558 SRX1137114 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846971: Fixed_RLT [US-1457350-9]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_Triton X Lysis|source_name;;H1 ECSs p40 Fixed Cells Triton X Lysis GEO Accession;;GSM1846971 GSM1846971 Fixed_RLT [US-1457350-9] 682500768 6691184 2015-10-05 11:31:16 428763035 682500768 6691184 2 6691184 index:0,count:6691184,average:51,stdev:0|index:1,count:6691184,average:51,stdev:0 GSM1846971_r1 24.28 1.47 0.06 565140288 848009270 487081231 774667406 150.05 159.04 6123811 5695986 186.206 1179.742 81 32927 78.03 91.11 9531917 4778322 9531917 4778322 81.36 87.31 9531917 4982201 9531917 4578813 24773602 4.38 1.49 0 13.14 0 0.19 0 0.05 0 0.00 0 8.24 0 6123811 0 102 0 99.88 0 1.33 0 0.03 0 1.14 0 0.01 0 253.56 0 0.30 0 99987 0 6691184 0 879283 0 12652 0 3271 0 0 0 551450 0 480 0 0 0 1962 0 369880 0 5466 0 377788 0 78.38 0 5244528 0 8733 381448 43.678919042712 6691184.0 6123811.0 99987.0 879283.0 12652.0 3271.0 0.0 551450.0 5244528.0 91.5 1.5 13.1 0.2 0.0 0.0 8.2 78.4 51 51 51.00 38 341250384 26.2 22.7 22.8 28.3 0.0 37.8 22.9 smartseq 573510 SRR2149808 SRP062177 SRS1028557 SRX1137115 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846972: Live_PLRC [US-1457351-10]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846972 GSM1846972 Live_PLRC [US-1457351-10] 858405786 8415743 2015-10-05 11:31:16 551460975 858405786 8415743 2 8415743 index:0,count:8415743,average:51,stdev:0|index:1,count:8415743,average:51,stdev:0 GSM1846972_r1 5.9 3.54 0.22 674533602 940079160 615964538 862125147 139.37 139.96 7166068 5709901 216.549 1767.841 87 30625 84.86 93.22 8183799 6081035 8183799 6081035 87.42 88.05 8183799 6264747 8183799 5743381 18414657 2.73 1.00 0 7.64 0 0.08 0 0.06 0 0.00 0 14.71 0 7166068 0 102 0 100.30 0 1.47 0 0.01 0 1.20 0 0.01 0 184.74 0 0.28 0 84396 0 8415743 0 642904 0 6653 0 5454 0 0 0 1237568 0 838 0 0 0 7098 0 1183248 0 7415 0 1198599 0 77.51 0 6523164 0 61911 1219717 19.701135500961 8415743.0 7166068.0 84396.0 642904.0 6653.0 5454.0 0.0 1237568.0 6523164.0 85.2 1.0 7.6 0.1 0.1 0.0 14.7 77.5 51 51 51.00 38 429202893 25.6 23.9 24.1 26.4 0.0 38.0 25.5 smartseq 573518 SRR2149809 SRP062177 SRS1028556 SRX1137116 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846973: Live_PLRC [US-1457351-11]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846973 GSM1846973 Live_PLRC [US-1457351-11] 851777316 8350758 2015-10-05 11:31:16 533774703 851777316 8350758 2 8350758 index:0,count:8350758,average:51,stdev:0|index:1,count:8350758,average:51,stdev:0 GSM1846973_r1 6.28 3.58 0.26 731982206 1013398950 672170402 933940307 138.45 138.94 7767552 6171312 219.798 1911.458 78 32155 83.84 91.57 8799885 6512412 8799885 6512412 86.37 86.92 8799885 6709204 8799885 6181448 25436373 3.47 1.15 0 7.85 0 0.11 0 0.08 0 0.00 0 6.79 0 7767552 0 102 0 100.28 0 1.46 0 0.01 0 1.22 0 0.01 0 227.75 0 0.25 0 95618 0 8350758 0 655733 0 9517 0 6985 0 0 0 566704 0 993 0 0 0 7884 0 1255434 0 7394 0 1271705 0 85.16 0 7111819 0 61752 1295839 20.984567301464 8350758.0 7767552.0 95618.0 655733.0 9517.0 6985.0 0.0 566704.0 7111819.0 93.0 1.1 7.9 0.1 0.1 0.0 6.8 85.2 51 51 51.00 38 425888658 25.8 23.6 23.7 26.8 0.0 38.1 25.4 smartseq 573583 SRR2149811 SRP062177 SRS1028554 SRX1137119 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846975: Live_PLRC [US-1457351-13]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI-|sample group;;Live cells_FRSCR|source_name;;H1 ESCs p40 Live Cells FRSCR GEO Accession;;GSM1846975 GSM1846975 Live_PLRC [US-1457351-13] 713515398 6995249 2015-10-05 11:31:16 444518338 713515398 6995249 2 6995249 index:0,count:6995249,average:51,stdev:0|index:1,count:6995249,average:51,stdev:0 GSM1846975_r1 6.11 3.62 0.25 610835239 846519961 561356107 780794468 138.58 139.09 6514952 5248692 210.278 1856.849 78 29043 85.47 93.29 7356705 5568142 7356705 5568142 87.74 88.4 7356705 5716241 7356705 5275960 16335927 2.67 1.16 0 7.81 0 0.09 0 0.08 0 0.00 0 6.70 0 6514952 0 102 0 100.26 0 1.43 0 0.01 0 1.22 0 0.01 0 273.73 0 0.24 0 81456 0 6995249 0 546450 0 5982 0 5487 0 0 0 468828 0 517 0 0 0 6970 0 1060513 0 5946 0 1073946 0 85.32 0 5968502 0 63044 1091931 17.320141488484 6995249.0 6514952.0 81456.0 546450.0 5982.0 5487.0 0.0 468828.0 5968502.0 93.1 1.2 7.8 0.1 0.1 0.0 6.7 85.3 51 51 51.00 38 356757699 26.0 23.4 23.5 27.1 0.0 38.1 25.6 smartseq 573591 SRR2149812 SRP062177 SRS1028553 SRX1137120 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846976: Fixed_PLRC [US-1457351-14]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846976 GSM1846976 Fixed_PLRC [US-1457351-14] 870597744 8535272 2015-10-05 11:31:16 547726076 870597744 8535272 2 8535272 index:0,count:8535272,average:51,stdev:0|index:1,count:8535272,average:51,stdev:0 GSM1846976_r1 7.3 3.34 0.28 706863897 976581138 643288537 892865182 138.16 138.8 7565382 6150856 210.515 1825.605 78 34181 81.59 89.96 8695949 6172664 8695949 6172664 84.4 84.88 8695949 6385329 8695949 5824175 29779884 4.21 1.17 0 8.25 0 0.11 0 0.09 0 0.00 0 11.17 0 7565382 0 102 0 100.11 0 1.83 0 0.02 0 1.19 0 0.01 0 138.41 0 0.28 0 99738 0 8535272 0 703986 0 9174 0 7526 0 0 0 953190 0 947 0 0 0 7005 0 1145781 0 11015 0 1164748 0 80.39 0 6861396 0 47482 1180313 24.858114653974 8535272.0 7565382.0 99738.0 703986.0 9174.0 7526.0 0.0 953190.0 6861396.0 88.6 1.2 8.2 0.1 0.1 0.0 11.2 80.4 51 51 51.00 38 435298872 25.9 23.6 23.6 27.0 0.0 38.0 25.1 smartseq 573599 SRR2149813 SRP062177 SRS1028552 SRX1137121 SRA282583 GEO Fixed single-cell transcriptomic characterization of human radial glial diversity The human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers. Overall design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR. GSM1846977: Fixed_PLRC [US-1457351-15]; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability). Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. Illumina HiSeq 2500 cell line;;H1|cell type;;cultured embryonic stem cells|phenotype;;DAPI+|sample group;;Fixed cells_FRSCR|source_name;;H1 ESCs p40 Fixed Cells FRSCR GEO Accession;;GSM1846977 GSM1846977 Fixed_PLRC [US-1457351-15] 686652270 6731885 2015-10-05 11:31:16 426807700 686652270 6731885 2 6731885 index:0,count:6731885,average:51,stdev:0|index:1,count:6731885,average:51,stdev:0 GSM1846977_r1 5.07 3.6 0.25 520553011 703652363 472461066 641683712 135.17 135.82 5659440 4774696 189.206 1574.044 78 29682 79.02 87.43 6572103 4472036 6572103 4472036 81.68 82.28 6572103 4622550 6572103 4208518 27670025 5.32 1.24 0 8.09 0 0.13 0 0.11 0 0.00 0 15.70 0 5659440 0 102 0 100.01 0 1.75 0 0.02 0 1.20 0 0.01 0 175.61 0 0.27 0 83586 0 6731885 0 544315 0 8475 0 7226 0 0 0 1056744 0 593 0 0 0 4977 0 825687 0 6925 0 838182 0 75.98 0 5115125 0 48539 848821 17.487401883022 6731885.0 5659440.0 83586.0 544315.0 8475.0 7226.0 0.0 1056744.0 5115125.0 84.1 1.2 8.1 0.1 0.1 0.0 15.7 76.0 51 51 51.00 38 343326135 25.8 23.5 23.5 27.2 0.0 38.0 24.7 smartseq 921881 SRR2558111 SRP064464 SRS1098849 SRX1304143 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901455: islet_single_cell_1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;undefined|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901455 GSM1901455 islet_single_cell_1 509805950 10196119 2016-01-05 16:18:03 280837927 509805950 10196119 1 10196119 index:0,count:10196119,average:50,stdev:0 GSM1901455_r1 2.14 1.38 0.06 391786899 448108470 275675204 364862516 114.38 132.35 0 0 0 0 0 0 64.77 92.64 20631668 5172491 20631668 5172491 72.15 87.48 20631668 5761682 20631668 4884406 14520744 3.71 4.35 0 23.56 0 0.39 0 0.13 0 0.00 0 21.16 0 7985402 0 50 0 49.38 0 1.46 0 0.01 0 1.14 0 0.01 0 412.43 0 0.47 0 443404 0 10196119 0 2402199 0 39728 0 13141 0 0 0 2157848 0 201 0 0 0 2563 0 395969 0 3816 0 402549 0 54.76 0 5583203 0 22321 445525 19.959903230142 10196119.0 7985402.0 443404.0 2402199.0 39728.0 13141.0 0.0 2157848.0 5583203.0 78.3 4.3 23.6 0.4 0.1 0.0 21.2 54.8 50 50 50.00 20 509805950 25.2 23.4 24.0 27.4 0.0 34.4 25.3 smartseq 921899 SRR2558112 SRP064464 SRS1098920 SRX1304144 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901456: islet_single_cell_2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901456 GSM1901456 islet_single_cell_2 357341400 7146828 2016-01-05 16:18:03 219230203 357341400 7146828 1 7146828 index:0,count:7146828,average:50,stdev:0 GSM1901456_r1 1.72 1.48 0.08 240716644 274099006 180247839 231057251 113.87 128.19 0 0 0 0 0 0 66.06 88.76 11392155 3242766 11392155 3242766 72.09 84.77 11392155 3538840 11392155 3097245 13857832 5.76 6.04 0 17.57 0 0.35 0 0.13 0 0.00 0 30.83 0 4909111 0 50 0 49.34 0 1.43 0 0.01 0 1.16 0 0.01 0 233.90 0 0.55 0 431633 0 7146828 0 1255610 0 25128 0 9193 0 0 0 2203396 0 99 0 0 0 1663 0 282960 0 2957 0 287679 0 51.12 0 3653501 0 32300 310002 9.597585139319 7146828.0 4909111.0 431633.0 1255610.0 25128.0 9193.0 0.0 2203396.0 3653501.0 68.7 6.0 17.6 0.4 0.1 0.0 30.8 51.1 50 50 50.00 20 357341400 25.9 22.9 23.7 27.6 0.0 33.4 24.3 smartseq 921914 SRR2558113 SRP064464 SRS1098919 SRX1304145 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901457: islet_single_cell_3; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;delta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901457 GSM1901457 islet_single_cell_3 687541950 13750839 2016-01-05 16:18:03 345119736 687541950 13750839 1 13750839 index:0,count:13750839,average:50,stdev:0 GSM1901457_r1 1.83 1.82 0.11 377400835 417098886 274478352 340847422 110.52 124.18 0 0 0 0 0 0 62.29 86.92 17721801 4849213 17721801 4849213 70.68 83.47 17721801 5502708 17721801 4656823 26659784 7.06 8.41 0 16.04 0 0.44 0 0.15 0 0.00 0 42.80 0 7785159 0 50 0 49.20 0 1.43 0 0.01 0 1.16 0 0.01 0 221.00 0 0.41 0 1156079 0 13750839 0 2206231 0 59928 0 20854 0 0 0 5884898 0 137 0 0 0 2173 0 293043 0 7265 0 302618 0 40.57 0 5578928 0 22244 328731 14.778412156087 13750839.0 7785159.0 1156079.0 2206231.0 59928.0 20854.0 0.0 5884898.0 5578928.0 56.6 8.4 16.0 0.4 0.2 0.0 42.8 40.6 50 50 50.00 20 687541950 25.3 22.5 23.9 28.3 0.0 35.3 26.5 smartseq 921930 SRR2558114 SRP064464 SRS1098918 SRX1304146 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901458: islet_single_cell_4; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901458 GSM1901458 islet_single_cell_4 414536900 8290738 2016-01-05 16:18:03 256680794 414536900 8290738 1 8290738 index:0,count:8290738,average:50,stdev:0 GSM1901458_r1 2.07 1.65 0.09 253893376 298218856 189327156 249306577 117.46 131.68 0 0 0 0 0 0 65.4 88.38 11776240 3393425 11776240 3393425 72.23 84.43 11776240 3747841 11776240 3241879 14587820 5.75 7.12 0 16.28 0 0.37 0 0.14 0 0.00 0 36.91 0 5188969 0 50 0 49.31 0 1.41 0 0.01 0 1.14 0 0.01 0 459.18 0 0.61 0 590130 0 8290738 0 1349403 0 30307 0 11526 0 0 0 3059936 0 178 0 0 0 1760 0 269821 0 3521 0 275280 0 46.31 0 3839566 0 31999 302605 9.456701771930 8290738.0 5188969.0 590130.0 1349403.0 30307.0 11526.0 0.0 3059936.0 3839566.0 62.6 7.1 16.3 0.4 0.1 0.0 36.9 46.3 50 50 50.00 20 414536900 25.7 22.8 23.8 27.6 0.0 33.2 24.0 smartseq 921945 SRR2558115 SRP064464 SRS1098916 SRX1304147 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901459: islet_single_cell_5; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;duct|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901459 GSM1901459 islet_single_cell_5 531737200 10634744 2016-01-05 16:18:03 271262585 531737200 10634744 1 10634744 index:0,count:10634744,average:50,stdev:0 GSM1901459_r1 2.42 1.6 0.18 386169142 466392924 281137499 383045193 120.77 136.25 0 0 0 0 0 0 66.12 91.41 18258256 5205583 18258256 5205583 73.57 86.03 18258256 5791559 18258256 4899195 16491653 4.27 5.07 0 20.48 0 0.37 0 0.17 0 0.00 0 25.43 0 7872610 0 50 0 49.37 0 1.38 0 0.01 0 1.15 0 0.01 0 364.62 0 0.41 0 539638 0 10634744 0 2177902 0 39675 0 17674 0 0 0 2704785 0 230 0 0 0 3060 0 466164 0 4558 0 474012 0 53.55 0 5694708 0 41968 527328 12.565001906214 10634744.0 7872610.0 539638.0 2177902.0 39675.0 17674.0 0.0 2704785.0 5694708.0 74.0 5.1 20.5 0.4 0.2 0.0 25.4 53.5 50 50 50.00 20 531737200 25.4 23.2 24.0 27.3 0.0 35.1 26.2 smartseq 921963 SRR2558116 SRP064464 SRS1098917 SRX1304148 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901460: islet_single_cell_6; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901460 GSM1901460 islet_single_cell_6 413631900 8272638 2016-01-05 16:18:03 256413434 413631900 8272638 1 8272638 index:0,count:8272638,average:50,stdev:0 GSM1901460_r1 1.54 1.67 0.12 286640011 336091472 223501187 288623695 117.25 129.14 0 0 0 0 0 0 67.6 87.35 12165016 3954595 12165016 3954595 73.36 83.6 12165016 4291554 12165016 3784586 19023589 6.64 5.73 0 15.99 0 0.39 0 0.18 0 0.00 0 28.72 0 5850147 0 50 0 49.37 0 1.45 0 0.01 0 1.14 0 0.01 0 263.55 0 0.59 0 473712 0 8272638 0 1323077 0 32056 0 14923 0 0 0 2375512 0 128 0 0 0 1880 0 293319 0 3576 0 298903 0 54.72 0 4527070 0 27211 327456 12.033956855683 8272638.0 5850147.0 473712.0 1323077.0 32056.0 14923.0 0.0 2375512.0 4527070.0 70.7 5.7 16.0 0.4 0.2 0.0 28.7 54.7 50 50 50.00 20 413631900 25.8 22.7 23.5 28.0 0.0 33.2 24.0 smartseq 921979 SRR2558117 SRP064464 SRS1098914 SRX1304149 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901461: islet_single_cell_7; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901461 GSM1901461 islet_single_cell_7 398954250 7979085 2016-01-05 16:18:03 235026606 398954250 7979085 1 7979085 index:0,count:7979085,average:50,stdev:0 GSM1901461_r1 1.06 0.8 0.08 337398608 375078046 214888360 281676064 111.17 131.08 0 0 0 0 0 0 60.83 95.71 19935264 4170473 19935264 4170473 73.3 91.66 19935264 5025682 19935264 3993892 6769085 2.01 2.99 0 31.32 0 0.36 0 0.10 0 0.00 0 13.61 0 6856128 0 50 0 49.32 0 1.40 0 0.00 0 1.14 0 0.00 0 305.58 0 0.56 0 238717 0 7979085 0 2498837 0 29121 0 7878 0 0 0 1085958 0 104 0 0 0 1856 0 515517 0 2907 0 520384 0 54.61 0 4357291 0 41303 698653 16.915308815340 7979085.0 6856128.0 238717.0 2498837.0 29121.0 7878.0 0.0 1085958.0 4357291.0 85.9 3.0 31.3 0.4 0.1 0.0 13.6 54.6 50 50 50.00 20 398954250 24.1 24.8 25.2 25.9 0.0 33.8 24.6 smartseq 921996 SRR2558118 SRP064464 SRS1098915 SRX1304150 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901462: islet_single_cell_8; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;delta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901462 GSM1901462 islet_single_cell_8 728473150 14569463 2016-01-05 16:18:03 366610145 728473150 14569463 1 14569463 index:0,count:14569463,average:50,stdev:0 GSM1901462_r1 1.83 1.53 0.08 467459444 515978807 347042190 429382602 110.38 123.73 0 0 0 0 0 0 64.61 87.9 22004347 6186749 22004347 6186749 71.7 84.39 22004347 6865212 22004347 5939577 29572148 6.33 6.72 0 17.41 0 0.40 0 0.16 0 0.00 0 33.71 0 9575502 0 50 0 49.31 0 1.49 0 0.01 0 1.16 0 0.01 0 359.25 0 0.41 0 978738 0 14569463 0 2536951 0 58881 0 23151 0 0 0 4911929 0 246 0 0 0 2713 0 407720 0 6631 0 417310 0 48.31 0 7038551 0 25443 455481 17.902016271666 14569463.0 9575502.0 978738.0 2536951.0 58881.0 23151.0 0.0 4911929.0 7038551.0 65.7 6.7 17.4 0.4 0.2 0.0 33.7 48.3 50 50 50.00 20 728473150 25.2 22.9 24.1 27.7 0.0 35.2 26.4 smartseq 922012 SRR2558119 SRP064464 SRS1098913 SRX1304151 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901463: islet_single_cell_9; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;undefined|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901463 GSM1901463 islet_single_cell_9 633170300 12663406 2016-01-05 16:18:03 322982463 633170300 12663406 1 12663406 index:0,count:12663406,average:50,stdev:0 GSM1901463_r1 1.56 1.96 0.07 385262170 453217320 294790102 380758180 117.64 129.16 0 0 0 0 0 0 67.29 88.87 16249008 5310499 16249008 5310499 74.29 84.73 16249008 5863163 16249008 5063063 24034263 6.24 7.23 0 15.13 0 0.37 0 0.16 0 0.00 0 37.15 0 7892198 0 50 0 49.33 0 1.42 0 0.01 0 1.17 0 0.01 0 272.98 0 0.39 0 915561 0 12663406 0 1916380 0 47124 0 20230 0 0 0 4703854 0 261 0 0 0 2980 0 395423 0 6110 0 404774 0 47.19 0 5975818 0 26329 451820 17.160545406206 12663406.0 7892198.0 915561.0 1916380.0 47124.0 20230.0 0.0 4703854.0 5975818.0 62.3 7.2 15.1 0.4 0.2 0.0 37.1 47.2 50 50 50.00 20 633170300 25.8 22.4 23.5 28.2 0.0 35.3 26.6 smartseq 922123 SRR2558120 SRP064464 SRS1098912 SRX1304152 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901464: islet_single_cell_10; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;duct|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901464 GSM1901464 islet_single_cell_10 408156450 8163129 2016-01-05 16:18:03 231656939 408156450 8163129 1 8163129 index:0,count:8163129,average:50,stdev:0 GSM1901464_r1 1.65 1.23 0.1 342204768 399360537 229879127 315703743 116.7 137.33 0 0 0 0 0 0 62.78 93.71 19025109 4358132 19025109 4358132 71.7 87.82 19025109 4977604 19025109 4083887 10224434 2.99 3.01 0 28.07 0 0.37 0 0.12 0 0.00 0 14.47 0 6941837 0 50 0 49.43 0 1.40 0 0.01 0 1.16 0 0.01 0 452.11 0 0.48 0 245309 0 8163129 0 2291283 0 30264 0 9531 0 0 0 1181497 0 238 0 0 0 2844 0 407791 0 3086 0 413959 0 56.97 0 4650554 0 40285 469836 11.662802531960 8163129.0 6941837.0 245309.0 2291283.0 30264.0 9531.0 0.0 1181497.0 4650554.0 85.0 3.0 28.1 0.4 0.1 0.0 14.5 57.0 50 50 50.00 20 408156450 25.1 24.0 24.5 26.4 0.0 34.3 25.2 smartseq 922139 SRR2558121 SRP064464 SRS1098910 SRX1304153 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901465: islet_single_cell_11; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901465 GSM1901465 islet_single_cell_11 646632700 12932654 2016-01-05 16:18:03 329333954 646632700 12932654 1 12932654 index:0,count:12932654,average:50,stdev:0 GSM1901465_r1 3.37 1.63 0.09 463181441 541684945 348887703 453744115 116.95 130.05 0 0 0 0 0 0 68.01 90.88 20879025 6422365 20879025 6422365 71.3 81.8 20879025 6733343 20879025 5780785 21435403 4.63 5.20 0 18.38 0 0.34 0 0.19 0 0.00 0 26.46 0 9443737 0 50 0 49.37 0 1.40 0 0.01 0 1.16 0 0.01 0 283.89 0 0.41 0 671882 0 12932654 0 2377143 0 43345 0 23994 0 0 0 3421578 0 212 0 0 0 3658 0 527507 0 5300 0 536677 0 54.64 0 7066594 0 34343 580743 16.910083568704 12932654.0 9443737.0 671882.0 2377143.0 43345.0 23994.0 0.0 3421578.0 7066594.0 73.0 5.2 18.4 0.3 0.2 0.0 26.5 54.6 50 50 50.00 20 646632700 25.5 23.0 23.9 27.6 0.0 35.1 26.3 smartseq 922156 SRR2558122 SRP064464 SRS1098911 SRX1304154 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901466: islet_single_cell_12; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901466 GSM1901466 islet_single_cell_12 484113700 9682274 2016-01-05 16:18:03 274052801 484113700 9682274 1 9682274 index:0,count:9682274,average:50,stdev:0 GSM1901466_r1 1.97 1.6 0.06 388913618 445350327 317378754 390562986 114.51 123.06 0 0 0 0 0 0 75.57 92.94 15187715 5971331 15187715 5971331 80.27 89.74 15187715 6342857 15187715 5766227 15274672 3.93 3.65 0 15.25 0 0.27 0 0.14 0 0.00 0 17.98 0 7902113 0 50 0 49.40 0 1.45 0 0.01 0 1.15 0 0.00 0 441.22 0 0.44 0 353740 0 9682274 0 1476882 0 25847 0 13362 0 0 0 1740952 0 131 0 0 0 2453 0 590026 0 3155 0 595765 0 66.36 0 6425231 0 30620 632621 20.660385369040 9682274.0 7902113.0 353740.0 1476882.0 25847.0 13362.0 0.0 1740952.0 6425231.0 81.6 3.7 15.3 0.3 0.1 0.0 18.0 66.4 50 50 50.00 20 484113700 26.4 22.7 23.1 27.7 0.0 34.3 25.3 smartseq 922170 SRR2558123 SRP064464 SRS1098909 SRX1304155 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901467: islet_single_cell_13; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901467 GSM1901467 islet_single_cell_13 660035750 13200715 2016-01-05 16:18:03 330649987 660035750 13200715 1 13200715 index:0,count:13200715,average:50,stdev:0 GSM1901467_r1 0.38 0.87 0.06 366506381 343627660 251228053 275925035 93.76 109.83 0 0 0 0 0 0 64.5 95.38 20965047 4863369 20965047 4863369 73.36 94.09 20965047 5531993 20965047 4797262 10918504 2.98 8.03 0 18.50 0 0.37 0 0.10 0 0.00 0 42.41 0 7540457 0 50 0 49.27 0 1.54 0 0.01 0 1.12 0 0.01 0 332.33 0 0.36 0 1059791 0 13200715 0 2441625 0 48810 0 13638 0 0 0 5597810 0 87 0 0 0 545 0 152105 0 4974 0 157711 0 38.63 0 5098832 0 3940 165256 41.943147208122 13200715.0 7540457.0 1059791.0 2441625.0 48810.0 13638.0 0.0 5597810.0 5098832.0 57.1 8.0 18.5 0.4 0.1 0.0 42.4 38.6 50 50 50.00 20 660035750 25.0 23.0 24.3 27.7 0.0 35.4 26.8 smartseq 922186 SRR2558124 SRP064464 SRS1098908 SRX1304156 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901468: islet_single_cell_14; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901468 GSM1901468 islet_single_cell_14 607941300 12158826 2016-01-05 16:18:03 296140109 607941300 12158826 1 12158826 index:0,count:12158826,average:50,stdev:0 GSM1901468_r1 2.59 1.77 0.27 337695694 339637408 251325780 277506036 100.57 110.42 0 0 0 0 0 0 51.24 69.83 15138076 3564949 15138076 3564949 59.39 67.55 15138076 4132526 15138076 3448578 41559965 12.31 8.12 0 15.23 0 0.49 0 0.24 0 0.00 0 42.05 0 6957727 0 50 0 49.23 0 1.39 0 0.01 0 1.14 0 0.01 0 282.40 0 0.39 0 987288 0 12158826 0 1852284 0 60116 0 28597 0 0 0 5112386 0 126 0 0 0 1215 0 213366 0 5110 0 219817 0 41.99 0 5105443 0 11529 238943 20.725388151618 12158826.0 6957727.0 987288.0 1852284.0 60116.0 28597.0 0.0 5112386.0 5105443.0 57.2 8.1 15.2 0.5 0.2 0.0 42.0 42.0 50 50 50.00 20 607941300 25.9 22.0 23.4 28.7 0.0 35.6 27.1 smartseq 922203 SRR2558125 SRP064464 SRS1098906 SRX1304157 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901469: islet_single_cell_15; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901469 GSM1901469 islet_single_cell_15 785659550 15713191 2016-01-05 16:18:03 395211117 785659550 15713191 1 15713191 index:0,count:15713191,average:50,stdev:0 GSM1901469_r1 1.57 0.87 0.06 433436246 397809668 241571946 280037979 91.78 115.92 0 0 0 0 0 0 44.06 79.89 31505085 3929895 31505085 3929895 51.0 67.9 31505085 4548763 31505085 3340186 30693353 7.08 8.32 0 25.45 0 0.48 0 0.17 0 0.00 0 42.59 0 8918852 0 50 0 49.11 0 1.43 0 0.01 0 1.15 0 0.01 0 441.93 0 0.43 0 1307148 0 15713191 0 3999466 0 75947 0 26667 0 0 0 6691725 0 174 0 0 0 1261 0 290505 0 6867 0 298807 0 31.31 0 4919386 0 11041 319171 28.907798206684 15713191.0 8918852.0 1307148.0 3999466.0 75947.0 26667.0 0.0 6691725.0 4919386.0 56.8 8.3 25.5 0.5 0.2 0.0 42.6 31.3 50 50 50.00 20 785659550 24.6 23.4 24.7 27.3 0.0 35.4 26.8 smartseq 922218 SRR2558126 SRP064464 SRS1098904 SRX1304158 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901470: islet_single_cell_16; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901470 GSM1901470 islet_single_cell_16 670778400 13415568 2016-01-05 16:18:03 328189793 670778400 13415568 1 13415568 index:0,count:13415568,average:50,stdev:0 GSM1901470_r1 4.85 1.91 0.11 462359983 546530264 375125327 470968269 118.2 125.55 0 0 0 0 0 0 63.81 79.43 16722623 6033879 16722623 6033879 70.46 76.8 16722623 6663014 16722623 5834316 52711459 11.40 5.86 0 13.86 0 0.40 0 0.26 0 0.00 0 28.86 0 9456373 0 50 0 49.38 0 1.41 0 0.01 0 1.16 0 0.01 0 235.59 0 0.32 0 786687 0 13415568 0 1859543 0 53540 0 34403 0 0 0 3871252 0 184 0 0 0 2083 0 454147 0 6202 0 462616 0 56.63 0 7596830 0 24215 489664 20.221515589511 13415568.0 9456373.0 786687.0 1859543.0 53540.0 34403.0 0.0 3871252.0 7596830.0 70.5 5.9 13.9 0.4 0.3 0.0 28.9 56.6 50 50 50.00 20 670778400 26.6 21.7 22.5 29.2 0.0 35.6 27.3 smartseq 922236 SRR2558127 SRP064464 SRS1098903 SRX1304159 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901471: islet_single_cell_17; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;NA|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;FALSE|source_name;;pancreatic islet GEO Accession;;GSM1901471 GSM1901471 islet_single_cell_17 789866250 15797325 2016-01-05 16:18:03 403215351 789866250 15797325 1 15797325 index:0,count:15797325,average:50,stdev:0 GSM1901471_r1 25.09 2.19 0.16 358008759 482452246 273843228 383459240 134.76 140.03 0 0 0 0 0 0 70.1 93.95 11493655 5235290 11493655 5235290 84.71 93.24 11493655 6326534 11493655 5195621 17993704 5.03 10.30 0 12.00 0 0.38 0 0.09 0 0.00 0 52.25 0 7468306 0 50 0 49.14 0 1.38 0 0.01 0 1.08 0 0.01 0 71.72 0 0.41 0 1627431 0 15797325 0 1896122 0 60427 0 13808 0 0 0 8254784 0 89 0 0 0 271 0 34462 0 8331 0 43153 0 35.27 0 5572184 0 2763 41460 15.005428881650 15797325.0 7468306.0 1627431.0 1896122.0 60427.0 13808.0 0.0 8254784.0 5572184.0 47.3 10.3 12.0 0.4 0.1 0.0 52.3 35.3 50 50 50.00 20 789866250 25.8 21.7 23.1 29.4 0.0 35.3 26.6 smartseq 922250 SRR2558128 SRP064464 SRS1098905 SRX1304160 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901472: islet_single_cell_18; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901472 GSM1901472 islet_single_cell_18 456767900 9135358 2016-01-05 16:18:03 230800833 456767900 9135358 1 9135358 index:0,count:9135358,average:50,stdev:0 GSM1901472_r1 0.69 1.06 0.09 395784595 457055021 259200005 351480977 115.48 135.6 0 0 0 0 0 0 62.37 95.45 22401519 5009046 22401519 5009046 73.4 89.99 22401519 5895262 22401519 4722349 7850368 1.98 2.50 0 30.48 0 0.38 0 0.13 0 0.00 0 11.57 0 8031800 0 50 0 49.39 0 1.40 0 0.01 0 1.16 0 0.00 0 265.22 0 0.38 0 228431 0 9135358 0 2784211 0 34534 0 11974 0 0 0 1057050 0 205 0 0 0 4060 0 556909 0 3204 0 564378 0 57.44 0 5247589 0 46810 732554 15.649519333476 9135358.0 8031800.0 228431.0 2784211.0 34534.0 11974.0 0.0 1057050.0 5247589.0 87.9 2.5 30.5 0.4 0.1 0.0 11.6 57.4 50 50 50.00 20 456767900 24.6 24.3 24.9 26.1 0.0 35.3 26.6 smartseq 922266 SRR2558129 SRP064464 SRS1098902 SRX1304161 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901473: islet_single_cell_19; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901473 GSM1901473 islet_single_cell_19 595431950 11908639 2016-01-05 16:18:03 294144405 595431950 11908639 1 11908639 index:0,count:11908639,average:50,stdev:0 GSM1901473_r1 2.67 1.79 0.07 467332225 566126301 350769252 468805841 121.14 133.65 0 0 0 0 0 0 68.44 91.7 20663687 6510397 20663687 6510397 76.2 87.21 20663687 7247929 20663687 6191201 19950045 4.27 4.02 0 20.26 0 0.36 0 0.16 0 0.00 0 19.60 0 9512047 0 50 0 49.41 0 1.42 0 0.01 0 1.16 0 0.01 0 529.27 0 0.35 0 478523 0 11908639 0 2412697 0 43147 0 19118 0 0 0 2334327 0 283 0 0 0 3668 0 580860 0 5335 0 590146 0 59.62 0 7099350 0 29458 662248 22.481091723810 11908639.0 9512047.0 478523.0 2412697.0 43147.0 19118.0 0.0 2334327.0 7099350.0 79.9 4.0 20.3 0.4 0.2 0.0 19.6 59.6 50 50 50.00 20 595431950 25.4 23.2 23.9 27.4 0.0 35.5 27.0 smartseq 922378 SRR2558130 SRP064464 SRS1098907 SRX1304162 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901474: islet_single_cell_20; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901474 GSM1901474 islet_single_cell_20 540117000 10802340 2016-01-05 16:18:03 286835979 540117000 10802340 1 10802340 index:0,count:10802340,average:50,stdev:0 GSM1901474_r1 2.27 1.8 0.13 363283625 427976697 283003984 364821881 117.81 128.91 0 0 0 0 0 0 67.89 87.88 15102868 5036222 15102868 5036222 74.25 84.09 15102868 5507850 15102868 4819213 23837002 6.56 6.08 0 15.62 0 0.37 0 0.19 0 0.00 0 30.76 0 7418301 0 50 0 49.38 0 1.46 0 0.01 0 1.15 0 0.01 0 268.20 0 0.42 0 656406 0 10802340 0 1687212 0 39916 0 21027 0 0 0 3323096 0 149 0 0 0 2503 0 365012 0 4722 0 372386 0 53.05 0 5731089 0 24339 409330 16.817864332964 10802340.0 7418301.0 656406.0 1687212.0 39916.0 21027.0 0.0 3323096.0 5731089.0 68.7 6.1 15.6 0.4 0.2 0.0 30.8 53.1 50 50 50.00 20 540117000 25.7 22.6 23.6 28.0 0.0 34.8 25.9 smartseq 922394 SRR2558131 SRP064464 SRS1098901 SRX1304163 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901475: islet_single_cell_21; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901475 GSM1901475 islet_single_cell_21 464982400 9299648 2016-01-05 16:18:03 231992231 464982400 9299648 1 9299648 index:0,count:9299648,average:50,stdev:0 GSM1901475_r1 1.71 1.44 0.05 426281406 486968600 346819855 427984887 114.24 123.4 0 0 0 0 0 0 76.28 93.94 16955979 6592399 16955979 6592399 80.57 90.17 16955979 6963210 16955979 6327868 13026858 3.06 1.56 0 17.47 0 0.25 0 0.17 0 0.00 0 6.65 0 8641997 0 50 0 49.42 0 1.40 0 0.01 0 1.17 0 0.00 0 423.78 0 0.32 0 145372 0 9299648 0 1624617 0 23006 0 15762 0 0 0 618883 0 208 0 0 0 2943 0 712031 0 2501 0 717683 0 75.46 0 7017380 0 29068 772018 26.559033989267 9299648.0 8641997.0 145372.0 1624617.0 23006.0 15762.0 0.0 618883.0 7017380.0 92.9 1.6 17.5 0.2 0.2 0.0 6.7 75.5 50 50 50.00 20 464982400 26.3 23.2 23.5 26.9 0.0 35.4 26.9 smartseq 922410 SRR2558132 SRP064464 SRS1098900 SRX1304164 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901476: islet_single_cell_22; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;duct|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901476 GSM1901476 islet_single_cell_22 432744950 8654899 2016-01-05 16:18:03 243510616 432744950 8654899 1 8654899 index:0,count:8654899,average:50,stdev:0 GSM1901476_r1 4.84 1.65 0.13 315024990 390591780 245901526 327331265 123.99 133.11 0 0 0 0 0 0 72.15 93.1 12261320 4632094 12261320 4632094 79.47 88.78 12261320 5102312 12261320 4417250 12842835 4.08 5.08 0 16.69 0 0.35 0 0.15 0 0.00 0 25.32 0 6420113 0 50 0 49.42 0 1.45 0 0.01 0 1.15 0 0.01 0 257.50 0 0.45 0 439444 0 8654899 0 1444451 0 30614 0 13069 0 0 0 2191103 0 117 0 0 0 1876 0 272561 0 3577 0 278131 0 57.49 0 4975662 0 22433 313461 13.973209111577 8654899.0 6420113.0 439444.0 1444451.0 30614.0 13069.0 0.0 2191103.0 4975662.0 74.2 5.1 16.7 0.4 0.2 0.0 25.3 57.5 50 50 50.00 20 432744950 26.2 22.5 23.2 28.2 0.0 34.4 25.5 smartseq 922426 SRR2558133 SRP064464 SRS1098899 SRX1304165 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901477: islet_single_cell_23; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;NA|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;FALSE|source_name;;pancreatic islet GEO Accession;;GSM1901477 GSM1901477 islet_single_cell_23 721241600 14424832 2016-01-05 16:18:03 377308024 721241600 14424832 1 14424832 index:0,count:14424832,average:50,stdev:0 GSM1901477_r1 2.19 3.09 0.23 199683189 196297429 158565512 160498618 98.3 101.22 0 0 0 0 0 0 68.97 90.27 6521323 2937543 6521323 2937543 78.86 90.08 6521323 3358725 6521323 2931335 15927749 7.98 13.35 0 6.97 0 0.42 0 0.10 0 0.00 0 69.96 0 4259128 0 50 0 48.73 0 1.57 0 0.01 0 1.16 0 0.01 0 172.52 0 0.53 0 1925641 0 14424832 0 1004954 0 59922 0 13898 0 0 0 10091884 0 87 0 0 0 191 0 19155 0 7865 0 27298 0 22.56 0 3254174 0 2676 18917 7.069133034380 14424832.0 4259128.0 1925641.0 1004954.0 59922.0 13898.0 0.0 10091884.0 3254174.0 29.5 13.3 7.0 0.4 0.1 0.0 70.0 22.6 50 50 50.00 20 721241600 25.4 22.0 23.6 28.9 0.0 34.8 25.8 smartseq 922442 SRR2558134 SRP064464 SRS1098897 SRX1304166 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901478: islet_single_cell_24; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;duct|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901478 GSM1901478 islet_single_cell_24 503863200 10077264 2016-01-05 16:18:03 248471844 503863200 10077264 1 10077264 index:0,count:10077264,average:50,stdev:0 GSM1901478_r1 2.31 1.44 0.12 422664578 512984097 306903457 419322580 121.37 136.63 0 0 0 0 0 0 67.61 93.41 20093206 5794286 20093206 5794286 75.42 87.93 20093206 6463426 20093206 5454168 13701575 3.24 2.96 0 23.49 0 0.33 0 0.14 0 0.00 0 14.48 0 8570380 0 50 0 49.48 0 1.38 0 0.01 0 1.17 0 0.01 0 315.46 0 0.35 0 298662 0 10077264 0 2367255 0 32824 0 14399 0 0 0 1459661 0 243 0 0 0 3748 0 518689 0 4133 0 526813 0 61.56 0 6203125 0 44966 592756 13.182315527287 10077264.0 8570380.0 298662.0 2367255.0 32824.0 14399.0 0.0 1459661.0 6203125.0 85.0 3.0 23.5 0.3 0.1 0.0 14.5 61.6 50 50 50.00 20 503863200 25.3 23.8 24.3 26.6 0.0 35.5 27.1 smartseq 922457 SRR2558135 SRP064464 SRS1098898 SRX1304167 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901479: islet_single_cell_25; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901479 GSM1901479 islet_single_cell_25 574672950 11493459 2016-01-05 16:18:03 296992214 574672950 11493459 1 11493459 index:0,count:11493459,average:50,stdev:0 GSM1901479_r1 2.12 1.34 0.07 440873824 509084674 318369605 422441056 115.47 132.69 0 0 0 0 0 0 64.31 89.57 22251051 5773045 22251051 5773045 71.71 85.47 22251051 6437281 22251051 5508803 22504965 5.10 4.36 0 22.03 0 0.37 0 0.20 0 0.00 0 21.32 0 8976926 0 50 0 49.40 0 1.47 0 0.01 0 1.18 0 0.01 0 435.54 0 0.40 0 500831 0 11493459 0 2531621 0 43023 0 23239 0 0 0 2450271 0 203 0 0 0 3101 0 506493 0 4354 0 514151 0 56.08 0 6445305 0 32051 556727 17.370035256310 11493459.0 8976926.0 500831.0 2531621.0 43023.0 23239.0 0.0 2450271.0 6445305.0 78.1 4.4 22.0 0.4 0.2 0.0 21.3 56.1 50 50 50.00 20 574672950 24.8 23.4 24.4 27.3 0.1 34.9 25.8 smartseq 922489 SRR2558137 SRP064464 SRS1098890 SRX1304169 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901481: islet_single_cell_27; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901481 GSM1901481 islet_single_cell_27 461668150 9233363 2016-01-05 16:18:03 240964783 461668150 9233363 1 9233363 index:0,count:9233363,average:50,stdev:0 GSM1901481_r1 1.16 0.92 0.08 414458587 487451645 291366025 382629661 117.61 131.32 0 0 0 0 0 0 67.32 95.95 20291254 5662335 20291254 5662335 79.2 92.23 20291254 6661486 20291254 5442578 8259341 1.99 1.95 0 27.18 0 0.29 0 0.13 0 0.00 0 8.48 0 8410890 0 50 0 49.37 0 1.39 0 0.00 0 1.13 0 0.00 0 415.50 0 0.41 0 179638 0 9233363 0 2509763 0 27074 0 12350 0 0 0 783049 0 135 0 0 0 3260 0 734713 0 3095 0 741203 0 63.91 0 5901127 0 42892 1015058 23.665438776462 9233363.0 8410890.0 179638.0 2509763.0 27074.0 12350.0 0.0 783049.0 5901127.0 91.1 1.9 27.2 0.3 0.1 0.0 8.5 63.9 50 50 50.00 20 461668150 24.3 24.5 25.0 26.1 0.1 34.9 25.9 smartseq 922505 SRR2558138 SRP064464 SRS1098889 SRX1304170 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901482: islet_single_cell_28; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901482 GSM1901482 islet_single_cell_28 506361200 10127224 2016-01-05 16:18:03 263608279 506361200 10127224 1 10127224 index:0,count:10127224,average:50,stdev:0 GSM1901482_r1 3.64 1.53 0.08 443840341 535737139 349966378 460062294 120.7 131.46 0 0 0 0 0 0 71.7 91.2 18271688 6449766 18271688 6449766 74.88 83.77 18271688 6736379 18271688 5924373 19462688 4.39 2.25 0 19.00 0 0.33 0 0.29 0 0.00 0 10.55 0 8995741 0 50 0 49.49 0 1.44 0 0.01 0 1.14 0 0.01 0 368.26 0 0.40 0 228222 0 10127224 0 1923941 0 32965 0 29666 0 0 0 1068852 0 186 0 0 0 3595 0 552520 0 3610 0 559911 0 69.83 0 7071800 0 38654 607696 15.721425984374 10127224.0 8995741.0 228222.0 1923941.0 32965.0 29666.0 0.0 1068852.0 7071800.0 88.8 2.3 19.0 0.3 0.3 0.0 10.6 69.8 50 50 50.00 20 506361200 25.6 23.3 23.7 27.3 0.1 34.9 25.8 smartseq 922521 SRR2558139 SRP064464 SRS1098888 SRX1304171 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901483: islet_single_cell_29; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;duct|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901483 GSM1901483 islet_single_cell_29 520206650 10404133 2016-01-05 16:18:03 272172910 520206650 10404133 1 10404133 index:0,count:10404133,average:50,stdev:0 GSM1901483_r1 3.04 1.59 0.13 427868894 524007393 312424948 428287726 122.47 137.08 0 0 0 0 0 0 69.61 95.67 19955972 6042621 19955972 6042621 77.85 90.4 19955972 6757491 19955972 5709487 8513550 1.99 3.22 0 22.72 0 0.34 0 0.15 0 0.00 0 16.08 0 8680373 0 50 0 49.46 0 1.41 0 0.01 0 1.15 0 0.01 0 271.41 0 0.40 0 335385 0 10404133 0 2364249 0 34942 0 15993 0 0 0 1672825 0 150 0 0 0 3158 0 497740 0 3595 0 504643 0 60.71 0 6316124 0 34611 571211 16.503741585045 10404133.0 8680373.0 335385.0 2364249.0 34942.0 15993.0 0.0 1672825.0 6316124.0 83.4 3.2 22.7 0.3 0.2 0.0 16.1 60.7 50 50 50.00 20 520206650 25.5 23.4 23.9 27.2 0.1 34.9 25.8 smartseq 922633 SRR2558140 SRP064464 SRS1098887 SRX1304172 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901484: islet_single_cell_30; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901484 GSM1901484 islet_single_cell_30 747877650 14957553 2016-01-05 16:18:03 379873287 747877650 14957553 1 14957553 index:0,count:14957553,average:50,stdev:0 GSM1901484_r1 0.66 2.35 0.14 275268492 272338983 201660763 216389197 98.94 107.3 0 0 0 0 0 0 63.35 88.86 11758081 3663448 11758081 3663448 74.24 87.59 11758081 4293205 11758081 3610947 19863842 7.22 11.65 0 11.10 0 0.43 0 0.13 0 0.00 0 60.78 0 5782757 0 50 0 48.92 0 1.54 0 0.01 0 1.11 0 0.01 0 297.50 0 0.46 0 1742076 0 14957553 0 1660232 0 64034 0 19604 0 0 0 9091158 0 81 0 0 0 643 0 166897 0 7824 0 175445 0 27.56 0 4122525 0 6183 179419 29.018114184053 14957553.0 5782757.0 1742076.0 1660232.0 64034.0 19604.0 0.0 9091158.0 4122525.0 38.7 11.6 11.1 0.4 0.1 0.0 60.8 27.6 50 50 50.00 20 747877650 25.4 21.8 23.5 29.3 0.1 35.0 25.8 smartseq 922649 SRR2558141 SRP064464 SRS1098886 SRX1304173 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901485: islet_single_cell_31; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;undefined|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901485 GSM1901485 islet_single_cell_31 704473350 14089467 2016-01-05 16:18:03 358501889 704473350 14089467 1 14089467 index:0,count:14089467,average:50,stdev:0 GSM1901485_r1 1.23 2.05 0.15 333205934 351884646 256612979 292593055 105.61 114.02 0 0 0 0 0 0 61.93 81.87 13466059 4264457 13466059 4264457 70.19 79.52 13466059 4833445 13466059 4142108 30983871 9.30 9.74 0 11.90 0 0.41 0 0.18 0 0.00 0 50.54 0 6885804 0 50 0 49.26 0 1.49 0 0.01 0 1.12 0 0.01 0 214.92 0 0.43 0 1372944 0 14089467 0 1676906 0 57239 0 25250 0 0 0 7121174 0 111 0 0 0 1243 0 157218 0 6563 0 165135 0 36.97 0 5208898 0 11137 184467 16.563437191344 14089467.0 6885804.0 1372944.0 1676906.0 57239.0 25250.0 0.0 7121174.0 5208898.0 48.9 9.7 11.9 0.4 0.2 0.0 50.5 37.0 50 50 50.00 20 704473350 25.5 22.1 23.4 29.0 0.1 35.0 25.8 smartseq 922667 SRR2558142 SRP064464 SRS1098885 SRX1304174 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901486: islet_single_cell_32; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901486 GSM1901486 islet_single_cell_32 661145650 13222913 2016-01-05 16:18:03 335107510 661145650 13222913 1 13222913 index:0,count:13222913,average:50,stdev:0 GSM1901486_r1 1.76 1.73 0.09 428179204 484963464 324498974 408349477 113.26 125.84 0 0 0 0 0 0 58.68 78.13 19106096 5136518 19106096 5136518 65.66 74.83 19106096 5746973 19106096 4919900 49443154 11.55 6.54 0 16.48 0 0.36 0 0.17 0 0.00 0 33.28 0 8752913 0 50 0 49.36 0 1.47 0 0.01 0 1.16 0 0.01 0 235.66 0 0.37 0 864466 0 13222913 0 2178506 0 47480 0 22590 0 0 0 4399930 0 170 0 0 0 2717 0 413672 0 5556 0 422115 0 49.72 0 6574407 0 23818 464095 19.485053321018 13222913.0 8752913.0 864466.0 2178506.0 47480.0 22590.0 0.0 4399930.0 6574407.0 66.2 6.5 16.5 0.4 0.2 0.0 33.3 49.7 50 50 50.00 20 661145650 26.1 22.2 23.2 28.5 0.1 35.1 26.1 smartseq 922685 SRR2558143 SRP064464 SRS1098884 SRX1304175 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901487: islet_single_cell_33; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901487 GSM1901487 islet_single_cell_33 591068300 11821366 2016-01-05 16:18:03 300969758 591068300 11821366 1 11821366 index:0,count:11821366,average:50,stdev:0 GSM1901487_r1 1.53 1.6 0.08 416725789 468134976 320375155 397578860 112.34 124.1 0 0 0 0 0 0 68.42 89.68 18341311 5823895 18341311 5823895 74.65 86.08 18341311 6354006 18341311 5590180 22610844 5.43 5.55 0 17.06 0 0.36 0 0.19 0 0.00 0 27.44 0 8511557 0 50 0 49.33 0 1.40 0 0.01 0 1.15 0 0.01 0 370.06 0 0.37 0 655941 0 11821366 0 2017143 0 42923 0 22661 0 0 0 3244225 0 233 0 0 0 2522 0 573218 0 4681 0 580654 0 54.94 0 6494414 0 33195 618595 18.635186021991 11821366.0 8511557.0 655941.0 2017143.0 42923.0 22661.0 0.0 3244225.0 6494414.0 72.0 5.5 17.1 0.4 0.2 0.0 27.4 54.9 50 50 50.00 20 591068300 25.7 22.7 23.4 28.1 0.1 35.1 26.0 smartseq 922701 SRR2558144 SRP064464 SRS1098883 SRX1304176 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901488: islet_single_cell_34; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901488 GSM1901488 islet_single_cell_34 466357000 9327140 2016-01-05 16:18:03 250551493 466357000 9327140 1 9327140 index:0,count:9327140,average:50,stdev:0 GSM1901488_r1 1.61 1.43 0.05 393404897 447080994 294933541 377798888 113.64 128.1 0 0 0 0 0 0 69.57 93.15 18727230 5559016 18727230 5559016 75.85 88.96 18727230 6060869 18727230 5309284 13616909 3.46 2.90 0 21.69 0 0.34 0 0.19 0 0.00 0 13.80 0 7990785 0 50 0 49.42 0 1.41 0 0.01 0 1.14 0 0.00 0 373.09 0 0.40 0 270021 0 9327140 0 2022749 0 31513 0 17813 0 0 0 1287029 0 277 0 0 0 2550 0 525433 0 3132 0 531392 0 63.99 0 5968036 0 28447 586244 20.608289099026 9327140.0 7990785.0 270021.0 2022749.0 31513.0 17813.0 0.0 1287029.0 5968036.0 85.7 2.9 21.7 0.3 0.2 0.0 13.8 64.0 50 50 50.00 20 466357000 25.6 23.2 23.7 27.4 0.0 34.7 25.6 smartseq 922718 SRR2558145 SRP064464 SRS1098882 SRX1304177 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901489: islet_single_cell_35; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901489 GSM1901489 islet_single_cell_35 535268950 10705379 2016-01-05 16:18:03 274771967 535268950 10705379 1 10705379 index:0,count:10705379,average:50,stdev:0 GSM1901489_r1 1.83 1.58 0.08 425276062 495516980 332794254 426256006 116.52 128.08 0 0 0 0 0 0 72.17 92.65 18123749 6240650 18123749 6240650 78.02 88.85 18123749 6746245 18123749 5984871 16302421 3.83 3.75 0 17.85 0 0.31 0 0.18 0 0.00 0 18.74 0 8647233 0 50 0 49.41 0 1.42 0 0.01 0 1.13 0 0.00 0 409.99 0 0.36 0 401612 0 10705379 0 1911308 0 33061 0 19105 0 0 0 2005980 0 263 0 0 0 3069 0 614524 0 3808 0 621664 0 62.92 0 6735925 0 37010 669825 18.098486895434 10705379.0 8647233.0 401612.0 1911308.0 33061.0 19105.0 0.0 2005980.0 6735925.0 80.8 3.8 17.9 0.3 0.2 0.0 18.7 62.9 50 50 50.00 20 535268950 26.1 22.7 23.4 27.8 0.1 35.1 26.1 smartseq 922734 SRR2558146 SRP064464 SRS1098881 SRX1304178 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901490: islet_single_cell_36; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901490 GSM1901490 islet_single_cell_36 527733800 10554676 2016-01-05 16:18:03 276145168 527733800 10554676 1 10554676 index:0,count:10554676,average:50,stdev:0 GSM1901490_r1 3.58 1.58 0.06 449979296 539115605 344447337 450350280 119.81 130.75 0 0 0 0 0 0 70.95 93.03 19212087 6476326 19212087 6476326 75.55 85.04 19212087 6896897 19212087 5919778 14865672 3.30 2.67 0 20.53 0 0.32 0 0.24 0 0.00 0 12.94 0 9128587 0 50 0 49.48 0 1.45 0 0.01 0 1.17 0 0.01 0 383.81 0 0.40 0 281662 0 10554676 0 2167326 0 34198 0 25630 0 0 0 1366261 0 188 0 0 0 4063 0 515197 0 3525 0 522973 0 65.95 0 6961261 0 31504 584731 18.560531995937 10554676.0 9128587.0 281662.0 2167326.0 34198.0 25630.0 0.0 1366261.0 6961261.0 86.5 2.7 20.5 0.3 0.2 0.0 12.9 66.0 50 50 50.00 20 527733800 25.2 23.5 24.0 27.2 0.1 34.9 25.8 smartseq 922751 SRR2558147 SRP064464 SRS1098880 SRX1304179 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901491: islet_single_cell_37; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;duct|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901491 GSM1901491 islet_single_cell_37 490211300 9804226 2016-01-05 16:18:03 252223999 490211300 9804226 1 9804226 index:0,count:9804226,average:50,stdev:0 GSM1901491_r1 2.51 1.33 0.14 398240232 472559881 272132128 375609358 118.66 138.02 0 0 0 0 0 0 64.31 94.48 21175285 5200356 21175285 5200356 73.52 88.78 21175285 5945193 21175285 4886495 10345256 2.60 3.48 0 26.34 0 0.37 0 0.15 0 0.00 0 16.99 0 8086607 0 50 0 49.44 0 1.38 0 0.01 0 1.16 0 0.01 0 107.94 0 0.37 0 340947 0 9804226 0 2582622 0 36454 0 15062 0 0 0 1666103 0 193 0 0 0 3245 0 464650 0 3652 0 471740 0 56.14 0 5503985 0 41474 535785 12.918575493080 9804226.0 8086607.0 340947.0 2582622.0 36454.0 15062.0 0.0 1666103.0 5503985.0 82.5 3.5 26.3 0.4 0.2 0.0 17.0 56.1 50 50 50.00 20 490211300 25.2 23.6 24.2 27.0 0.1 35.1 26.1 smartseq 922765 SRR2558148 SRP064464 SRS1098879 SRX1304180 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901492: islet_single_cell_38; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901492 GSM1901492 islet_single_cell_38 528430300 10568606 2016-01-05 16:18:03 269099069 528430300 10568606 1 10568606 index:0,count:10568606,average:50,stdev:0 GSM1901492_r1 2.42 1.47 0.09 397989174 470236021 290663867 388828134 118.15 133.77 0 0 0 0 0 0 64.59 88.98 19074514 5233861 19074514 5233861 72.25 84.48 19074514 5854146 19074514 4969253 21421507 5.38 4.54 0 21.01 0 0.38 0 0.19 0 0.00 0 22.76 0 8103129 0 50 0 49.41 0 1.42 0 0.01 0 1.14 0 0.01 0 98.82 0 0.36 0 480164 0 10568606 0 2220911 0 40131 0 19605 0 0 0 2405741 0 276 0 0 0 3071 0 439586 0 4131 0 447064 0 55.66 0 5882218 0 35297 495956 14.050939173301 10568606.0 8103129.0 480164.0 2220911.0 40131.0 19605.0 0.0 2405741.0 5882218.0 76.7 4.5 21.0 0.4 0.2 0.0 22.8 55.7 50 50 50.00 20 528430300 25.3 23.0 24.0 27.6 0.1 35.2 26.2 smartseq 922783 SRR2558149 SRP064464 SRS1098878 SRX1304181 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901493: islet_single_cell_39; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901493 GSM1901493 islet_single_cell_39 533938600 10678772 2016-01-05 16:18:03 271075903 533938600 10678772 1 10678772 index:0,count:10678772,average:50,stdev:0 GSM1901493_r1 1.61 1.29 0.07 451074986 517333616 325138067 429793182 114.69 132.19 0 0 0 0 0 0 65.34 90.96 23218320 5982979 23218320 5982979 72.11 86.35 23218320 6602975 23218320 5679653 19986136 4.43 2.88 0 24.15 0 0.35 0 0.20 0 0.00 0 13.70 0 9156375 0 50 0 49.43 0 1.40 0 0.01 0 1.16 0 0.01 0 346.34 0 0.34 0 307403 0 10678772 0 2578722 0 37576 0 21362 0 0 0 1463459 0 228 0 0 0 3388 0 599642 0 3505 0 606763 0 61.60 0 6577653 0 41879 667384 15.936006112849 10678772.0 9156375.0 307403.0 2578722.0 37576.0 21362.0 0.0 1463459.0 6577653.0 85.7 2.9 24.1 0.4 0.2 0.0 13.7 61.6 50 50 50.00 20 533938600 25.6 23.3 23.9 27.2 0.1 35.2 26.3 smartseq 922894 SRR2558150 SRP064464 SRS1098877 SRX1304182 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901494: islet_single_cell_40; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;duct|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901494 GSM1901494 islet_single_cell_40 523441350 10468827 2016-01-05 16:18:03 268733491 523441350 10468827 1 10468827 index:0,count:10468827,average:50,stdev:0 GSM1901494_r1 2.21 1.32 0.1 453990444 546248444 324997505 447280929 120.32 137.63 0 0 0 0 0 0 67.02 93.87 22625751 6165498 22625751 6165498 74.33 87.94 22625751 6837298 22625751 5776019 13843544 3.05 2.42 0 25.13 0 0.32 0 0.16 0 0.00 0 11.64 0 9199070 0 50 0 49.48 0 1.35 0 0.01 0 1.16 0 0.01 0 376.88 0 0.37 0 253399 0 10468827 0 2631172 0 33554 0 17112 0 0 0 1219091 0 314 0 0 0 4187 0 578895 0 3626 0 587022 0 62.74 0 6567898 0 47649 661143 13.875275451741 10468827.0 9199070.0 253399.0 2631172.0 33554.0 17112.0 0.0 1219091.0 6567898.0 87.9 2.4 25.1 0.3 0.2 0.0 11.6 62.7 50 50 50.00 20 523441350 25.3 23.7 24.3 26.7 0.1 35.1 26.1 smartseq 922910 SRR2558151 SRP064464 SRS1098876 SRX1304183 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901495: islet_single_cell_41; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901495 GSM1901495 islet_single_cell_41 579662950 11593259 2016-01-05 16:18:03 293671289 579662950 11593259 1 11593259 index:0,count:11593259,average:50,stdev:0 GSM1901495_r1 2.08 1.77 0.07 431501247 516782743 331177968 436031044 119.76 131.66 0 0 0 0 0 0 67.89 88.98 18433533 5961142 18433533 5961142 74.38 84.39 18433533 6531276 18433533 5653613 23831492 5.52 4.74 0 17.95 0 0.36 0 0.20 0 0.00 0 23.69 0 8781045 0 50 0 49.43 0 1.43 0 0.01 0 1.15 0 0.01 0 293.91 0 0.35 0 549092 0 11593259 0 2081330 0 42032 0 23588 0 0 0 2746594 0 234 0 0 0 3662 0 507423 0 4289 0 515608 0 57.79 0 6699715 0 33323 575909 17.282627614561 11593259.0 8781045.0 549092.0 2081330.0 42032.0 23588.0 0.0 2746594.0 6699715.0 75.7 4.7 18.0 0.4 0.2 0.0 23.7 57.8 50 50 50.00 20 579662950 25.7 22.8 23.5 27.9 0.1 35.2 26.3 smartseq 922925 SRR2558152 SRP064464 SRS1098875 SRX1304184 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901496: islet_single_cell_42; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901496 GSM1901496 islet_single_cell_42 520710650 10414213 2016-01-05 16:18:03 266188553 520710650 10414213 1 10414213 index:0,count:10414213,average:50,stdev:0 GSM1901496_r1 3.44 1.65 0.08 441313919 529630892 349396894 455616472 120.01 130.4 0 0 0 0 0 0 71.62 90.82 17870229 6412308 17870229 6412308 74.83 83.53 17870229 6699244 17870229 5897078 20632838 4.68 2.78 0 18.18 0 0.34 0 0.28 0 0.00 0 13.41 0 8953034 0 50 0 49.49 0 1.41 0 0.01 0 1.16 0 0.01 0 337.76 0 0.36 0 289871 0 10414213 0 1892821 0 35914 0 28643 0 0 0 1396622 0 251 0 0 0 3574 0 522973 0 4212 0 531010 0 67.79 0 7060213 0 34651 575056 16.595653805085 10414213.0 8953034.0 289871.0 1892821.0 35914.0 28643.0 0.0 1396622.0 7060213.0 86.0 2.8 18.2 0.3 0.3 0.0 13.4 67.8 50 50 50.00 20 520710650 25.7 23.1 23.6 27.6 0.1 35.1 26.2 smartseq 922940 SRR2558153 SRP064464 SRS1098874 SRX1304185 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901497: islet_single_cell_43; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901497 GSM1901497 islet_single_cell_43 504460300 10089206 2016-01-05 16:18:03 257347662 504460300 10089206 1 10089206 index:0,count:10089206,average:50,stdev:0 GSM1901497_r1 1.69 1.37 0.07 445350918 509364154 361270035 447371255 114.37 123.83 0 0 0 0 0 0 74.13 91.64 17845171 6695834 17845171 6695834 78.69 88.14 17845171 7107500 17845171 6440382 20412424 4.58 2.15 0 17.10 0 0.32 0 0.22 0 0.00 0 9.93 0 9032683 0 50 0 49.44 0 1.43 0 0.01 0 1.15 0 0.01 0 359.62 0 0.33 0 217287 0 10089206 0 1725709 0 32534 0 22281 0 0 0 1001708 0 183 0 0 0 2592 0 678186 0 3066 0 684027 0 72.42 0 7306974 0 23701 727474 30.693810387747 10089206.0 9032683.0 217287.0 1725709.0 32534.0 22281.0 0.0 1001708.0 7306974.0 89.5 2.2 17.1 0.3 0.2 0.0 9.9 72.4 50 50 50.00 20 504460300 26.3 22.9 23.3 27.4 0.1 35.1 26.2 smartseq 922956 SRR2558154 SRP064464 SRS1098873 SRX1304186 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901498: islet_single_cell_44; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901498 GSM1901498 islet_single_cell_44 647464350 12949287 2016-01-05 16:18:03 330422536 647464350 12949287 1 12949287 index:0,count:12949287,average:50,stdev:0 GSM1901498_r1 0.08 0.89 0.06 390095484 382577287 247559783 284533539 98.07 114.94 0 0 0 0 0 0 59.72 95.33 23425844 4802801 23425844 4802801 72.09 93.36 23425844 5797631 23425844 4703434 11486936 2.94 7.52 0 23.20 0 0.42 0 0.14 0 0.00 0 37.33 0 8041823 0 50 0 49.14 0 1.52 0 0.01 0 1.11 0 0.01 0 200.94 0 0.41 0 973948 0 12949287 0 3003647 0 54754 0 18127 0 0 0 4834583 0 79 0 0 0 792 0 376278 0 5988 0 383137 0 38.91 0 5038176 0 5761 514772 89.354625932998 12949287.0 8041823.0 973948.0 3003647.0 54754.0 18127.0 0.0 4834583.0 5038176.0 62.1 7.5 23.2 0.4 0.1 0.0 37.3 38.9 50 50 50.00 20 647464350 24.5 23.3 24.3 27.9 0.1 35.1 26.1 smartseq 922972 SRR2558155 SRP064464 SRS1098872 SRX1304187 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901499: islet_single_cell_45; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901499 GSM1901499 islet_single_cell_45 674298850 13485977 2016-01-05 16:18:03 341210405 674298850 13485977 1 13485977 index:0,count:13485977,average:50,stdev:0 GSM1901499_r1 3.53 1.81 0.13 427513599 498842509 345782747 431171822 116.68 124.69 0 0 0 0 0 0 66.8 83.55 15622330 5851480 15622330 5851480 73.61 81.05 15622330 6448129 15622330 5676554 38811135 9.08 6.83 0 13.02 0 0.39 0 0.19 0 0.00 0 34.47 0 8759601 0 50 0 49.37 0 1.51 0 0.01 0 1.15 0 0.01 0 274.29 0 0.37 0 921349 0 13485977 0 1756005 0 52382 0 25957 0 0 0 4648037 0 170 0 0 0 1790 0 344138 0 5859 0 351957 0 51.93 0 7003596 0 14502 379533 26.171079851055 13485977.0 8759601.0 921349.0 1756005.0 52382.0 25957.0 0.0 4648037.0 7003596.0 65.0 6.8 13.0 0.4 0.2 0.0 34.5 51.9 50 50 50.00 20 674298850 25.9 21.9 23.1 29.0 0.1 35.2 26.2 smartseq 922988 SRR2558156 SRP064464 SRS1098871 SRX1304188 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901500: islet_single_cell_46; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;NA|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;FALSE|source_name;;pancreatic islet GEO Accession;;GSM1901500 GSM1901500 islet_single_cell_46 679736050 13594721 2016-01-05 16:18:03 345292008 679736050 13594721 1 13594721 index:0,count:13594721,average:50,stdev:0 GSM1901500_r1 1.98 2.58 0.17 292323762 290285151 238117071 242652326 99.3 101.9 0 0 0 0 0 0 74.0 93.66 9261676 4542269 9261676 4542269 83.74 93.37 9261676 5140461 9261676 4528246 16363561 5.60 10.76 0 9.48 0 0.45 0 0.09 0 0.00 0 54.31 0 6138576 0 50 0 49.10 0 1.57 0 0.01 0 1.09 0 0.01 0 282.90 0 0.41 0 1462891 0 13594721 0 1288730 0 60992 0 12493 0 0 0 7382660 0 71 0 0 0 268 0 21224 0 7528 0 29091 0 35.67 0 4849846 0 2047 22079 10.786028334148 13594721.0 6138576.0 1462891.0 1288730.0 60992.0 12493.0 0.0 7382660.0 4849846.0 45.2 10.8 9.5 0.4 0.1 0.0 54.3 35.7 50 50 50.00 20 679736050 24.9 21.7 22.9 30.4 0.1 35.0 25.8 smartseq 923004 SRR2558157 SRP064464 SRS1098870 SRX1304189 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901501: islet_single_cell_47; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;NA|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;FALSE|source_name;;pancreatic islet GEO Accession;;GSM1901501 GSM1901501 islet_single_cell_47 683738250 13674765 2016-01-05 16:18:03 350243167 683738250 13674765 1 13674765 index:0,count:13674765,average:50,stdev:0 GSM1901501_r1 0.01 2.24 0.15 375974676 365957941 330331944 326725548 97.34 98.91 0 0 0 0 0 0 81.99 95.66 10103403 6409799 10103403 6409799 88.89 95.64 10103403 6948526 10103403 6408567 16125941 4.29 8.56 0 8.16 0 0.43 0 0.10 0 0.00 0 42.31 0 7817388 0 50 0 49.30 0 1.56 0 0.01 0 1.12 0 0.01 0 199.31 0 0.37 0 1170561 0 13674765 0 1116450 0 58839 0 13063 0 0 0 5785475 0 54 0 0 0 149 0 14508 0 6900 0 21611 0 49.00 0 6700938 0 1777 12607 7.094541361846 13674765.0 7817388.0 1170561.0 1116450.0 58839.0 13063.0 0.0 5785475.0 6700938.0 57.2 8.6 8.2 0.4 0.1 0.0 42.3 49.0 50 50 50.00 20 683738250 25.2 21.6 22.6 30.6 0.1 35.0 25.8 smartseq 923021 SRR2558158 SRP064464 SRS1098869 SRX1304190 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901502: islet_single_cell_48; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901502 GSM1901502 islet_single_cell_48 496968600 9939372 2016-01-05 16:18:03 256242726 496968600 9939372 1 9939372 index:0,count:9939372,average:50,stdev:0 GSM1901502_r1 1.54 1.14 0.08 426720114 539675510 330553914 430151048 126.47 130.13 0 0 0 0 0 0 73.79 95.67 15109482 6414860 15109482 6414860 86.67 93.0 15109482 7534091 15109482 6235547 10261684 2.40 2.69 0 20.00 0 0.27 0 0.16 0 0.00 0 12.12 0 8693090 0 50 0 49.30 0 1.37 0 0.00 0 1.15 0 0.00 0 225.04 0 0.39 0 267495 0 9939372 0 1987902 0 26353 0 15656 0 0 0 1204273 0 123 0 0 0 1810 0 841016 0 3474 0 846423 0 67.46 0 6705188 0 20645 1248991 60.498474206830 9939372.0 8693090.0 267495.0 1987902.0 26353.0 15656.0 0.0 1204273.0 6705188.0 87.5 2.7 20.0 0.3 0.2 0.0 12.1 67.5 50 50 50.00 20 496968600 23.7 24.7 25.5 26.1 0.1 35.1 26.1 smartseq 923036 SRR2558159 SRP064464 SRS1098868 SRX1304191 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901503: islet_single_cell_49; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;NA|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;FALSE|source_name;;pancreatic islet GEO Accession;;GSM1901503 GSM1901503 islet_single_cell_49 1595428500 31908570 2016-01-05 16:18:03 844694679 1595428500 31908570 1 31908570 index:0,count:31908570,average:50,stdev:0 GSM1901503_r1 0.02 2.16 0.14 877587927 855273992 776508857 768550238 97.46 98.98 0 0 0 0 0 0 82.74 95.73 23286865 15076058 23286865 15076058 89.21 95.7 23286865 16255714 23286865 15071419 36363500 4.14 8.46 0 7.75 0 0.42 0 0.10 0 0.00 0 42.37 0 18221293 0 50 0 49.31 0 1.66 0 0.01 0 1.10 0 0.01 0 256.41 0 0.45 0 2700085 0 31908570 0 2473208 0 135445 0 33447 0 0 0 13518385 0 141 0 0 0 333 0 34673 0 15933 0 51080 0 49.35 0 15748085 0 4297 41411 9.637188736328 31908570.0 18221293.0 2700085.0 2473208.0 135445.0 33447.0 0.0 13518385.0 15748085.0 57.1 8.5 7.8 0.4 0.1 0.0 42.4 49.4 50 50 50.00 20 1595428500 25.1 21.7 22.7 30.4 0.1 34.5 25.1 smartseq 923149 SRR2558160 SRP064464 SRS1098867 SRX1304192 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901504: islet_single_cell_50; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;pp|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901504 GSM1901504 islet_single_cell_50 991633300 19832666 2016-01-05 16:18:03 533390734 991633300 19832666 1 19832666 index:0,count:19832666,average:50,stdev:0 GSM1901504_r1 3.54 2.17 0.14 756459130 935482310 628556252 816980663 123.67 129.98 0 0 0 0 0 0 73.61 89.26 25427647 11352812 25427647 11352812 79.41 85.57 25427647 12246720 25427647 10883261 45404866 6.00 4.46 0 13.63 0 0.38 0 0.24 0 0.00 0 21.62 0 15422954 0 50 0 49.42 0 1.41 0 0.01 0 1.15 0 0.01 0 303.82 0 0.45 0 883898 0 19832666 0 2703701 0 75497 0 47253 0 0 0 4286962 0 366 0 0 0 6254 0 872657 0 8236 0 887513 0 64.13 0 12719253 0 21892 976184 44.590900785675 19832666.0 15422954.0 883898.0 2703701.0 75497.0 47253.0 0.0 4286962.0 12719253.0 77.8 4.5 13.6 0.4 0.2 0.0 21.6 64.1 50 50 50.00 20 991633300 25.3 22.8 23.6 28.2 0.1 34.5 25.2 smartseq 923163 SRR2558161 SRP064464 SRS1098866 SRX1304193 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901505: islet_single_cell_51; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;undefined|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901505 GSM1901505 islet_single_cell_51 1373300550 27466011 2016-01-05 16:18:03 720364278 1373300550 27466011 1 27466011 index:0,count:27466011,average:50,stdev:0 GSM1901505_r1 1.57 2.5 0.13 1036294658 1253255160 878859764 1101655929 120.94 125.35 0 0 0 0 0 0 75.56 89.85 32467245 15967121 32467245 15967121 80.55 85.85 32467245 17021968 32467245 15256071 62403678 6.02 4.58 0 12.24 0 0.35 0 0.20 0 0.00 0 22.52 0 21132224 0 50 0 49.45 0 1.49 0 0.01 0 1.17 0 0.01 0 270.16 0 0.41 0 1257807 0 27466011 0 3361188 0 94996 0 53722 0 0 0 6185069 0 462 0 0 0 7586 0 1062127 0 11881 0 1082056 0 64.70 0 17771036 0 18657 1194611 64.030176341320 27466011.0 21132224.0 1257807.0 3361188.0 94996.0 53722.0 0.0 6185069.0 17771036.0 76.9 4.6 12.2 0.3 0.2 0.0 22.5 64.7 50 50 50.00 20 1373300550 25.8 22.4 23.0 28.7 0.1 34.8 25.5 smartseq 923180 SRR2558162 SRP064464 SRS1098865 SRX1304194 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901506: islet_single_cell_52; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901506 GSM1901506 islet_single_cell_52 1285905850 25718117 2016-01-05 16:18:03 690877116 1285905850 25718117 1 25718117 index:0,count:25718117,average:50,stdev:0 GSM1901506_r1 5.36 2.25 0.09 964849060 1203590241 810953447 1043602387 124.74 128.69 0 0 0 0 0 0 74.72 89.52 29738169 14677244 29738169 14677244 79.31 83.74 29738169 15577658 29738169 13728273 57511702 5.96 4.57 0 12.63 0 0.36 0 0.23 0 0.00 0 23.03 0 19641929 0 50 0 49.46 0 1.46 0 0.01 0 1.15 0 0.01 0 331.85 0 0.45 0 1175197 0 25718117 0 3247030 0 93646 0 59744 0 0 0 5922798 0 550 0 0 0 7411 0 981355 0 11562 0 1000878 0 63.75 0 16394899 0 20902 1113255 53.260692756674 25718117.0 19641929.0 1175197.0 3247030.0 93646.0 59744.0 0.0 5922798.0 16394899.0 76.4 4.6 12.6 0.4 0.2 0.0 23.0 63.7 50 50 50.00 20 1285905850 25.9 22.4 23.0 28.6 0.1 34.5 25.3 smartseq 923211 SRR2558164 SRP064464 SRS1098863 SRX1304196 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901508: islet_single_cell_54; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;duct|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901508 GSM1901508 islet_single_cell_54 1545240300 30904806 2016-01-05 16:18:03 815961012 1545240300 30904806 1 30904806 index:0,count:30904806,average:50,stdev:0 GSM1901508_r1 3.1 2.22 0.18 1143294715 1454619393 943810135 1254931710 127.23 132.96 0 0 0 0 0 0 75.26 91.82 37578739 17527740 37578739 17527740 80.92 86.74 37578739 18845940 37578739 16556602 53913567 4.72 4.81 0 13.60 0 0.34 0 0.20 0 0.00 0 24.10 0 23290799 0 50 0 49.44 0 1.39 0 0.01 0 1.14 0 0.01 0 340.24 0 0.44 0 1485719 0 30904806 0 4202096 0 105790 0 61500 0 0 0 7446717 0 667 0 0 0 7688 0 1321754 0 12789 0 1342898 0 61.77 0 19088703 0 25274 1492653 59.058835166574 30904806.0 23290799.0 1485719.0 4202096.0 105790.0 61500.0 0.0 7446717.0 19088703.0 75.4 4.8 13.6 0.3 0.2 0.0 24.1 61.8 50 50 50.00 20 1545240300 25.6 22.8 23.5 28.1 0.1 34.6 25.3 smartseq 923227 SRR2558165 SRP064464 SRS1098862 SRX1304197 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901509: islet_single_cell_55; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901509 GSM1901509 islet_single_cell_55 1233820700 24676414 2016-01-05 16:18:03 652272173 1233820700 24676414 1 24676414 index:0,count:24676414,average:50,stdev:0 GSM1901509_r1 6.87 2.07 0.14 818722770 981560150 664672105 826405293 119.89 124.33 0 0 0 0 0 0 70.95 88.63 26950364 11940773 26950364 11940773 76.64 82.36 26950364 12898504 26950364 11095679 53670299 6.56 6.55 0 13.61 0 0.44 0 0.22 0 0.00 0 31.14 0 16830689 0 50 0 49.34 0 1.47 0 0.01 0 1.16 0 0.01 0 281.12 0 0.46 0 1616083 0 24676414 0 3358201 0 108601 0 53888 0 0 0 7683236 0 235 0 0 0 3939 0 639907 0 12196 0 656277 0 54.60 0 13472488 0 11527 706353 61.278129608745 24676414.0 16830689.0 1616083.0 3358201.0 108601.0 53888.0 0.0 7683236.0 13472488.0 68.2 6.5 13.6 0.4 0.2 0.0 31.1 54.6 50 50 50.00 20 1233820700 25.0 22.4 23.0 29.6 0.1 34.6 25.2 smartseq 923243 SRR2558166 SRP064464 SRS1098861 SRX1304198 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901510: islet_single_cell_56; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901510 GSM1901510 islet_single_cell_56 994665200 19893304 2016-01-05 16:18:03 527449084 994665200 19893304 1 19893304 index:0,count:19893304,average:50,stdev:0 GSM1901510_r1 2.32 1.35 0.14 877162754 1102247104 694252408 891344070 125.66 128.39 0 0 0 0 0 0 75.68 96.0 29226349 13507383 29226349 13507383 87.6 92.68 29226349 15634849 29226349 13040685 17546747 2.00 2.28 0 19.00 0 0.27 0 0.18 0 0.00 0 9.83 0 17848865 0 50 0 49.34 0 1.38 0 0.00 0 1.13 0 0.00 0 378.92 0 0.44 0 453244 0 19893304 0 3778831 0 52984 0 34954 0 0 0 1956501 0 220 0 0 0 4925 0 1740209 0 7631 0 1752985 0 70.73 0 14070034 0 29622 2450330 82.719937884005 19893304.0 17848865.0 453244.0 3778831.0 52984.0 34954.0 0.0 1956501.0 14070034.0 89.7 2.3 19.0 0.3 0.2 0.0 9.8 70.7 50 50 50.00 20 994665200 24.4 24.1 24.6 26.9 0.1 34.7 25.5 smartseq 923259 SRR2558167 SRP064464 SRS1098860 SRX1304199 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901511: islet_single_cell_57; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901511 GSM1901511 islet_single_cell_57 914297000 18285940 2016-01-05 16:18:03 484413331 914297000 18285940 1 18285940 index:0,count:18285940,average:50,stdev:0 GSM1901511_r1 1.24 1.85 0.17 705289659 848885285 549189896 691726522 120.36 125.95 0 0 0 0 0 0 75.56 97.82 25988782 10889702 25988782 10889702 83.75 91.49 25988782 12069377 25988782 10184976 12494585 1.77 4.35 0 17.93 0 0.36 0 0.13 0 0.00 0 20.70 0 14411867 0 50 0 49.33 0 1.46 0 0.01 0 1.13 0 0.00 0 361.70 0 0.46 0 795796 0 18285940 0 3278925 0 65274 0 24373 0 0 0 3784426 0 341 0 0 0 4413 0 1013532 0 7975 0 1026261 0 60.88 0 11132942 0 13552 1303115 96.156655844156 18285940.0 14411867.0 795796.0 3278925.0 65274.0 24373.0 0.0 3784426.0 11132942.0 78.8 4.4 17.9 0.4 0.1 0.0 20.7 60.9 50 50 50.00 20 914297000 24.7 23.3 23.8 28.1 0.1 34.6 25.3 smartseq 923275 SRR2558168 SRP064464 SRS1098859 SRX1304200 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901512: islet_single_cell_58; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901512 GSM1901512 islet_single_cell_58 810995500 16219910 2016-01-05 16:18:03 428901157 810995500 16219910 1 16219910 index:0,count:16219910,average:50,stdev:0 GSM1901512_r1 2.67 2.56 0.2 700440629 929166234 576339083 789795001 132.65 137.04 0 0 0 0 0 0 76.78 93.7 22217962 10920109 22217962 10920109 81.99 86.21 22217962 11661375 22217962 10047998 24710885 3.53 2.54 0 15.83 0 0.30 0 0.21 0 0.00 0 11.80 0 14222954 0 50 0 49.45 0 1.40 0 0.01 0 1.17 0 0.01 0 535.70 0 0.43 0 412145 0 16219910 0 2568365 0 49150 0 33610 0 0 0 1914196 0 307 0 0 0 6077 0 1032650 0 6905 0 1045939 0 71.85 0 11654589 0 36999 1252625 33.855644747155 16219910.0 14222954.0 412145.0 2568365.0 49150.0 33610.0 0.0 1914196.0 11654589.0 87.7 2.5 15.8 0.3 0.2 0.0 11.8 71.9 50 50 50.00 20 810995500 25.4 23.3 23.7 27.6 0.1 34.7 25.5 smartseq 923291 SRR2558169 SRP064464 SRS1098858 SRX1304201 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901513: islet_single_cell_59; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901513 GSM1901513 islet_single_cell_59 1307074900 26141498 2016-01-05 16:18:03 691624162 1307074900 26141498 1 26141498 index:0,count:26141498,average:50,stdev:0 GSM1901513_r1 2.45 1.7 0.11 1128113882 1379949763 834714102 1100977243 122.32 131.9 0 0 0 0 0 0 66.6 90.67 47345465 15351809 47345465 15351809 76.83 85.57 47345465 17709913 47345465 14487750 54446272 4.83 2.79 0 23.41 0 0.49 0 0.26 0 0.00 0 11.07 0 23051251 0 50 0 49.30 0 1.43 0 0.01 0 1.13 0 0.01 0 482.61 0 0.46 0 728119 0 26141498 0 6120515 0 127383 0 68087 0 0 0 2894777 0 200 0 0 0 6977 0 1276192 0 12150 0 1295519 0 64.77 0 16930736 0 17036 1654161 97.097969006809 26141498.0 23051251.0 728119.0 6120515.0 127383.0 68087.0 0.0 2894777.0 16930736.0 88.2 2.8 23.4 0.5 0.3 0.0 11.1 64.8 50 50 50.00 20 1307074900 24.9 23.0 23.5 28.5 0.1 34.7 25.4 smartseq 923404 SRR2558170 SRP064464 SRS1098857 SRX1304202 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901514: islet_single_cell_60; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;NA|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;FALSE|source_name;;pancreatic islet GEO Accession;;GSM1901514 GSM1901514 islet_single_cell_60 1394085500 27881710 2016-01-05 16:18:03 737342445 1394085500 27881710 1 27881710 index:0,count:27881710,average:50,stdev:0 GSM1901514_r1 0.82 2.33 0.15 780462378 769503901 677309973 678618547 98.6 100.19 0 0 0 0 0 0 80.86 95.54 21418160 13128965 21418160 13128965 88.45 95.42 21418160 14360506 21418160 13112848 33778350 4.33 8.45 0 8.95 0 0.46 0 0.11 0 0.00 0 41.20 0 16236200 0 50 0 49.29 0 1.56 0 0.01 0 1.13 0 0.01 0 393.62 0 0.44 0 2355499 0 27881710 0 2494102 0 128163 0 30370 0 0 0 11486977 0 116 0 0 0 425 0 43523 0 15549 0 59613 0 49.29 0 13742098 0 5182 44401 8.568313392513 27881710.0 16236200.0 2355499.0 2494102.0 128163.0 30370.0 0.0 11486977.0 13742098.0 58.2 8.4 8.9 0.5 0.1 0.0 41.2 49.3 50 50 50.00 20 1394085500 25.0 21.4 22.2 31.3 0.1 34.6 25.2 smartseq 923419 SRR2558171 SRP064464 SRS1098856 SRX1304203 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901515: islet_single_cell_61; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901515 GSM1901515 islet_single_cell_61 815382000 16307640 2016-01-05 16:18:03 615278048 815382000 16307640 1 16307640 index:0,count:16307640,average:50,stdev:0 GSM1901515_r1 1.08 1.28 0.03 734236354 838399474 536317240 707064567 114.19 131.84 0 0 0 0 0 0 69.03 94.63 37694215 10278687 37694215 10278687 74.91 89.81 37694215 11154371 37694215 9755016 19342982 2.63 1.87 0 24.70 0 0.31 0 0.09 0 0.00 0 8.30 0 14889406 0 50 0 49.38 0 1.48 0 0.01 0 1.17 0 0.00 0 510.50 0 0.64 0 304266 0 16307640 0 4027484 0 50160 0 15263 0 0 0 1352811 0 338 0 0 0 5964 0 959231 0 4337 0 969870 0 66.61 0 10861922 0 27278 1073127 39.340384192389 16307640.0 14889406.0 304266.0 4027484.0 50160.0 15263.0 0.0 1352811.0 10861922.0 91.3 1.9 24.7 0.3 0.1 0.0 8.3 66.6 50 50 50.00 38 815382000 25.2 24.3 25.1 25.4 0.0 34.1 20.5 smartseq 923435 SRR2558172 SRP064464 SRS1098855 SRX1304204 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901516: islet_single_cell_62; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901516 GSM1901516 islet_single_cell_62 595434600 11908692 2016-01-05 16:18:03 448121287 595434600 11908692 1 11908692 index:0,count:11908692,average:50,stdev:0 GSM1901516_r1 1.53 1.19 0.09 499443801 555423436 389887082 482216762 111.21 123.68 0 0 0 0 0 0 63.19 81.24 22396909 6417323 22396909 6417323 68.05 78.07 22396909 6910792 22396909 6166652 48789331 9.77 3.00 0 18.95 0 0.37 0 0.19 0 0.00 0 14.16 0 10155171 0 50 0 49.36 0 1.46 0 0.01 0 1.14 0 0.01 0 354.31 0 0.63 0 357251 0 11908692 0 2256322 0 44356 0 23189 0 0 0 1685976 0 178 0 0 0 2759 0 547704 0 3532 0 554173 0 66.33 0 7898849 0 20656 594299 28.771252904725 11908692.0 10155171.0 357251.0 2256322.0 44356.0 23189.0 0.0 1685976.0 7898849.0 85.3 3.0 18.9 0.4 0.2 0.0 14.2 66.3 50 50 50.00 38 595434600 26.8 22.4 23.4 27.4 0.0 34.2 20.8 smartseq 923452 SRR2558173 SRP064464 SRS1098854 SRX1304205 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901517: islet_single_cell_63; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901517 GSM1901517 islet_single_cell_63 700729750 14014595 2016-01-05 16:18:03 515877567 700729750 14014595 1 14014595 index:0,count:14014595,average:50,stdev:0 GSM1901517_r1 2.48 1.38 0.06 626137364 738809602 471133321 614269425 117.99 130.38 0 0 0 0 0 0 69.07 92.09 28325206 8780297 28325206 8780297 75.68 86.81 28325206 9620633 28325206 8277123 25642399 4.10 1.91 0 22.68 0 0.37 0 0.15 0 0.00 0 8.78 0 12712975 0 50 0 49.41 0 1.49 0 0.01 0 1.16 0 0.01 0 403.62 0 0.55 0 267287 0 14014595 0 3178648 0 51307 0 20397 0 0 0 1229916 0 178 0 0 0 5529 0 729725 0 4603 0 740035 0 68.03 0 9534327 0 23333 823020 35.272789611280 14014595.0 12712975.0 267287.0 3178648.0 51307.0 20397.0 0.0 1229916.0 9534327.0 90.7 1.9 22.7 0.4 0.1 0.0 8.8 68.0 50 50 50.00 38 700729750 25.9 23.2 23.9 27.0 0.0 34.7 21.4 smartseq 923469 SRR2558174 SRP064464 SRS1098853 SRX1304206 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901518: islet_single_cell_64; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901518 GSM1901518 islet_single_cell_64 240000700 4800014 2016-01-05 16:18:03 198707938 240000700 4800014 1 4800014 index:0,count:4800014,average:50,stdev:0 GSM1901518_r1 3.17 1.36 0.11 175487282 202220119 126148571 166484634 115.23 131.98 0 0 0 0 0 0 63.73 89.05 8925881 2282617 8925881 2282617 70.53 84.12 8925881 2526402 8925881 2156389 9403467 5.36 5.19 0 21.22 0 0.43 0 0.16 0 0.00 0 24.78 0 3581911 0 50 0 49.21 0 1.47 0 0.00 0 1.14 0 0.01 0 233.51 0 1.62 0 248938 0 4800014 0 1018492 0 20589 0 7920 0 0 0 1189594 0 54 0 0 0 1103 0 156708 0 1240 0 159105 0 53.40 0 2563419 0 26569 174072 6.551695585080 4800014.0 3581911.0 248938.0 1018492.0 20589.0 7920.0 0.0 1189594.0 2563419.0 74.6 5.2 21.2 0.4 0.2 0.0 24.8 53.4 50 50 50.00 38 240000700 26.8 21.5 25.6 26.1 0.0 29.7 17.1 smartseq 923483 SRR2558175 SRP064464 SRS1098852 SRX1304207 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901519: islet_single_cell_65; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901519 GSM1901519 islet_single_cell_65 757176400 15143528 2016-01-05 16:18:03 558860127 757176400 15143528 1 15143528 index:0,count:15143528,average:50,stdev:0 GSM1901519_r1 2.62 1.35 0.08 661928794 768023003 486168555 643408376 116.03 132.34 0 0 0 0 0 0 62.88 85.92 32528657 8459495 32528657 8459495 68.19 79.83 32528657 9174462 32528657 7860011 44890069 6.78 2.31 0 23.83 0 0.45 0 0.29 0 0.00 0 10.42 0 13453582 0 50 0 49.38 0 1.46 0 0.01 0 1.16 0 0.01 0 413.01 0 0.56 0 349060 0 15143528 0 3608126 0 68480 0 43933 0 0 0 1577533 0 277 0 0 0 4900 0 694346 0 5227 0 704750 0 65.01 0 9845456 0 40741 776420 19.057460543433 15143528.0 13453582.0 349060.0 3608126.0 68480.0 43933.0 0.0 1577533.0 9845456.0 88.8 2.3 23.8 0.5 0.3 0.0 10.4 65.0 50 50 50.00 38 757176400 26.0 23.0 23.9 27.2 0.0 34.7 21.3 smartseq 923498 SRR2558176 SRP064464 SRS1098851 SRX1304208 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901520: islet_single_cell_66; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901520 GSM1901520 islet_single_cell_66 693722000 13874440 2016-01-05 16:18:03 513592215 693722000 13874440 1 13874440 index:0,count:13874440,average:50,stdev:0 GSM1901520_r1 5.78 2.31 0.2 614199380 790227809 518776176 689259046 128.66 132.86 0 0 0 0 0 0 77.95 92.61 19099418 9720382 19099418 9720382 82.62 87.13 19099418 10302421 19099418 9144595 24689412 4.02 2.08 0 14.23 0 0.27 0 0.15 0 0.00 0 9.70 0 12470286 0 50 0 49.43 0 1.42 0 0.01 0 1.16 0 0.01 0 537.08 0 0.59 0 287917 0 13874440 0 1974579 0 37676 0 20945 0 0 0 1345533 0 285 0 0 0 6157 0 744512 0 5125 0 756079 0 75.65 0 10495707 0 21542 836452 38.828892396249 13874440.0 12470286.0 287917.0 1974579.0 37676.0 20945.0 0.0 1345533.0 10495707.0 89.9 2.1 14.2 0.3 0.2 0.0 9.7 75.6 50 50 50.00 38 693722000 25.7 23.3 24.1 26.8 0.0 34.6 21.3 smartseq 923514 SRR2558177 SRP064464 SRS1098850 SRX1304209 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901521: islet_single_cell_67; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;acinar|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901521 GSM1901521 islet_single_cell_67 423119900 8462398 2016-01-05 16:18:03 347283997 423119900 8462398 1 8462398 index:0,count:8462398,average:50,stdev:0 GSM1901521_r1 7.74 1.1 0.12 332067540 405157896 249687921 326188960 122.01 130.64 0 0 0 0 0 0 71.97 96.33 13914224 4883892 13914224 4883892 81.94 92.95 13914224 5560468 13914224 4712843 7337023 2.21 4.13 0 20.27 0 0.43 0 0.08 0 0.00 0 19.30 0 6785919 0 50 0 49.25 0 1.48 0 0.01 0 1.11 0 0.01 0 84.39 0 1.47 0 349646 0 8462398 0 1715716 0 36760 0 6532 0 0 0 1633187 0 84 0 0 0 1154 0 210932 0 2361 0 214531 0 59.91 0 5070203 0 7053 277312 39.318304267688 8462398.0 6785919.0 349646.0 1715716.0 36760.0 6532.0 0.0 1633187.0 5070203.0 80.2 4.1 20.3 0.4 0.1 0.0 19.3 59.9 50 50 50.00 38 423119900 25.3 23.0 25.6 26.1 0.0 30.2 17.3 smartseq 923530 SRR2558178 SRP064464 SRS1098896 SRX1304210 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901522: islet_single_cell_68; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901522 GSM1901522 islet_single_cell_68 530606400 10612128 2016-01-05 16:18:03 418781375 530606400 10612128 1 10612128 index:0,count:10612128,average:50,stdev:0 GSM1901522_r1 0.65 1.76 0.17 441532199 480617287 397173891 442149387 108.85 111.32 0 0 0 0 0 0 79.94 89.48 12419816 7216699 12419816 7216699 83.09 87.65 12419816 7500678 12419816 7068594 28290736 6.41 3.22 0 9.07 0 0.40 0 0.16 0 0.00 0 14.37 0 9027110 0 50 0 49.25 0 1.48 0 0.01 0 1.12 0 0.01 0 626.29 0 0.96 0 341776 0 10612128 0 962145 0 42848 0 17010 0 0 0 1525160 0 151 0 0 0 1484 0 494672 0 3377 0 499684 0 76.00 0 8064965 0 8188 525057 64.125183194919 10612128.0 9027110.0 341776.0 962145.0 42848.0 17010.0 0.0 1525160.0 8064965.0 85.1 3.2 9.1 0.4 0.2 0.0 14.4 76.0 50 50 50.00 38 530606400 27.4 21.5 22.8 28.2 0.0 32.4 19.1 smartseq 923546 SRR2558179 SRP064464 SRS1098895 SRX1304211 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901523: islet_single_cell_69; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;beta|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901523 GSM1901523 islet_single_cell_69 758112300 15162246 2016-01-05 16:18:03 567700786 758112300 15162246 1 15162246 index:0,count:15162246,average:50,stdev:0 GSM1901523_r1 3.97 2.17 0.1 643189568 795120676 546607064 700507203 123.62 128.16 0 0 0 0 0 0 74.64 88.18 20517552 9750166 20517552 9750166 78.23 83.06 20517552 10219302 20517552 9183597 43170870 6.71 2.70 0 13.24 0 0.34 0 0.23 0 0.00 0 13.28 0 13063441 0 50 0 49.44 0 1.48 0 0.01 0 1.16 0 0.01 0 352.16 0 0.63 0 409553 0 15162246 0 2006802 0 51158 0 34813 0 0 0 2012834 0 416 0 0 0 5332 0 700047 0 5772 0 711567 0 72.92 0 11056639 0 20597 794981 38.596931591979 15162246.0 13063441.0 409553.0 2006802.0 51158.0 34813.0 0.0 2012834.0 11056639.0 86.2 2.7 13.2 0.3 0.2 0.0 13.3 72.9 50 50 50.00 38 758112300 26.2 23.1 23.8 27.0 0.0 34.3 21.0 smartseq 923658 SRR2558180 SRP064464 SRS1098894 SRX1304212 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901524: islet_single_cell_70; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;alpha|cell count;;1|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901524 GSM1901524 islet_single_cell_70 651900500 13038010 2016-01-05 16:18:03 486915261 651900500 13038010 1 13038010 index:0,count:13038010,average:50,stdev:0 GSM1901524_r1 0.67 0.91 0.11 513327257 526380792 445371246 474460100 102.54 106.53 0 0 0 0 0 0 80.44 93.47 17290357 8426277 17290357 8426277 84.56 92.69 17290357 8858174 17290357 8355615 18456153 3.60 3.66 0 11.20 0 0.35 0 0.16 0 0.00 0 19.15 0 10475184 0 50 0 49.40 0 1.52 0 0.01 0 1.19 0 0.01 0 366.69 0 0.62 0 477510 0 13038010 0 1460356 0 45518 0 20675 0 0 0 2496633 0 21 0 0 0 878 0 201739 0 3734 0 206372 0 69.14 0 9014828 0 3204 231905 72.379837702871 13038010.0 10475184.0 477510.0 1460356.0 45518.0 20675.0 0.0 2496633.0 9014828.0 80.3 3.7 11.2 0.3 0.2 0.0 19.1 69.1 50 50 50.00 38 651900500 25.6 23.1 24.0 27.3 0.0 34.3 20.8 smartseq 923674 SRR2558181 SRP064464 SRS1098893 SRX1304213 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901525: islet_500_cells_1; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;islet|cell count;;500|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901525 GSM1901525 islet_500_cells_1 1584267350 31685347 2016-01-05 16:18:03 868612974 1584267350 31685347 1 31685347 index:0,count:31685347,average:50,stdev:0 GSM1901525_r1 4.56 1.89 0.13 1463000687 1852777204 1153216670 1552500850 126.64 134.62 0 0 0 0 0 0 65.92 84.03 54540353 19617492 54540353 19617492 73.5 79.48 54540353 21873100 54540353 18557052 122075118 8.34 1.70 0 20.24 0 0.44 0 0.29 0 0.00 0 5.35 0 29759936 0 50 0 49.39 0 1.39 0 0.01 0 1.18 0 0.01 0 438.72 0 0.46 0 539942 0 31685347 0 6412732 0 140999 0 90365 0 0 0 1694047 0 438 0 0 0 7229 0 1653250 0 11499 0 1672416 0 73.68 0 23347204 0 96389 2002567 20.775887290043 31685347.0 29759936.0 539942.0 6412732.0 140999.0 90365.0 0.0 1694047.0 23347204.0 93.9 1.7 20.2 0.4 0.3 0.0 5.3 73.7 50 50 50.00 20 1584267350 26.8 22.0 22.5 28.7 0.0 34.3 25.4 smartseq 923688 SRR2558182 SRP064464 SRS1098892 SRX1304214 SRA302692 GEO Single-cell transcriptomics reveals unique features of human pancreatic islet cell subtypes We report the single-cell RNA-seq based identification of 6 known human islet cell types (alpha cells, beta cells, delta cells, pp cells, acinar cells and duct cells) based on the expression of known marker genes. We further assess cell type specific gene expression and suggest novel marker genes for several cell types. Overall design: Transcriptional dissection of human pancreatic islets of one donor using single-cell RNA-seq GSM1901526: islet_500_cells_2; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA single To disassociate islets into single cells, islets were incubated in Accutase (Life technology) in 37 °C for 20 min, neutralized by CMRL medium. Purification of single cells was performed by flow cytometry cell sorting on a Moflo AstriosEQ (Beckman Coulter, Miami) cDNA synthesis and enrichment was performed following the Smart-seq2 protocol as described (Picelli et al., 2014). Illumina HiSeq 2000 assigned cell type;;islet|cell count;;500|ercc_dilution;;1000000|ercc_mix;;mix2|qc pass;;TRUE|source_name;;pancreatic islet GEO Accession;;GSM1901526 GSM1901526 islet_500_cells_2 1342934550 26858691 2016-01-05 16:18:03 756559895 1342934550 26858691 1 26858691 index:0,count:26858691,average:50,stdev:0 GSM1901526_r1 12.44 2.32 0.18 1247458583 1720366287 1044853185 1465986811 137.91 140.31 0 0 0 0 0 0 71.59 85.77 35886062 18114916 35886062 18114916 79.32 81.67 35886062 20071067 35886062 17247705 97722069 7.83 1.50 0 15.58 0 0.35 0 0.25 0 0.00 0 5.20 0 25303079 0 50 0 49.47 0 1.40 0 0.01 0 1.18 0 0.01 0 57.80 0 0.48 0 401582 0 26858691 0 4183699 0 93813 0 66466 0 0 0 1395333 0 377 0 0 0 6064 0 1364844 0 8422 0 1379707 0 78.63 0 21119380 0 91984 1601075 17.406016263698 26858691.0 25303079.0 401582.0 4183699.0 93813.0 66466.0 0.0 1395333.0 21119380.0 94.2 1.5 15.6 0.3 0.2 0.0 5.2 78.6 50 50 50.00 20 1342934550 27.3 21.8 22.1 28.9 0.0 34.0 25.1 smartseq 1441809 SRR4251000 SRP090061 SRS1698679 SRX2171001 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317219: 54Dn2_G10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317219 GSM2317219 54Dn2_G10_smart-seq 160586900 1605869 2016-09-30 15:56:31 70309502 160586900 1605869 2 1605869 index:0,count:1605869,average:50,stdev:0|index:1,count:1605869,average:50,stdev:0 GSM2317219_r1 7.1 3.15 0.14 137217607 184717461 128777641 175023362 134.62 135.91 1456255 1174377 243.359 2318.742 100 5136 78.19 83.54 1694393 1138670 1694393 1138670 78.83 79.92 1694393 1147922 1694393 1089352 12912918 9.41 1.82 0 5.81 0 0.09 0 0.17 0 0.00 0 9.05 0 1456255 0 100 0 98.70 0 1.30 0 0.01 0 1.17 0 0.01 0 206.47 0 0.19 0 29241 0 1605869 0 93279 0 1473 0 2755 0 0 0 145386 0 107 0 0 0 1394 0 183363 0 1049 0 185913 0 84.87 0 1362976 0 37444 187478 5.006890289499 1605869.0 1456255.0 29241.0 93279.0 1473.0 2755.0 0.0 145386.0 1362976.0 90.7 1.8 5.8 0.1 0.2 0.0 9.1 84.9 50 50 50.00 24 80293450 26.5 22.2 22.3 29.0 0.0 36.8 24.4 smartseq 1441810 SRR4252000 SRP090061 SRS1699677 SRX2172001 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318219: SK_2_C09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318219 GSM2318219 SK_2_C09_smart-seq 40910000 409100 2016-09-30 15:56:31 28656722 40910000 409100 2 409100 index:0,count:409100,average:50,stdev:0|index:1,count:409100,average:50,stdev:0 GSM2318219_r1 0.21 4.84 0.54 777485 557553 696294 505048 71.71 72.53 10552 9305 101.173 12954.261 51 413 9.28 10.58 13628 979 13628 979 10.52 10.42 13628 1110 13628 964 299428 38.51 0.27 0 0.32 0 0.02 0 0.02 0 0.00 0 97.39 0 10552 0 100 0 90.43 0 1.15 0 0.01 0 1.04 0 0.00 0 52.60 0 0.50 0 1116 0 409100 0 1299 0 62 0 64 0 0 0 398422 0 0 0 0 0 1 0 73 0 32 0 106 0 2.26 0 9253 0 38 58 1.526315789474 409100.0 10552.0 1116.0 1299.0 62.0 64.0 0.0 398422.0 9253.0 2.6 0.3 0.3 0.0 0.0 0.0 97.4 2.3 50 50 50.00 38 20455000 22.1 22.5 21.9 33.5 0.0 35.2 15.7 smartseq 1441840 SRR4252001 SRP090061 SRS1699678 SRX2172002 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318220: SK_2_C10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318220 GSM2318220 SK_2_C10_smart-seq 239972200 2399722 2016-09-30 15:56:31 166786289 239972200 2399722 2 2399722 index:0,count:2399722,average:50,stdev:0|index:1,count:2399722,average:50,stdev:0 GSM2318220_r1 3.4 2.87 0.15 201815522 255463994 192723482 245359485 126.58 127.31 2175050 1886274 209.350 1719.977 81 9022 68.94 72.32 2428849 1499576 2428849 1499576 68.39 68.92 2428849 1487492 2428849 1429145 32477625 16.09 1.76 0 4.23 0 0.08 0 0.10 0 0.00 0 9.18 0 2175050 0 100 0 98.62 0 1.38 0 0.01 0 1.22 0 0.01 0 359.96 0 0.22 0 42330 0 2399722 0 101409 0 1826 0 2447 0 0 0 220399 0 99 0 0 0 1961 0 193851 0 1674 0 197585 0 86.41 0 2073641 0 33361 199764 5.987950001499 2399722.0 2175050.0 42330.0 101409.0 1826.0 2447.0 0.0 220399.0 2073641.0 90.6 1.8 4.2 0.1 0.1 0.0 9.2 86.4 50 50 50.00 38 119986100 27.3 21.8 21.9 29.1 0.0 37.3 23.9 smartseq 1441874 SRR4251002 SRP090061 SRS1698680 SRX2171003 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317221: 54Dn2_H01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317221 GSM2317221 54Dn2_H01_smart-seq 124191900 1241919 2016-09-30 15:56:31 56416858 124191900 1241919 2 1241919 index:0,count:1241919,average:50,stdev:0|index:1,count:1241919,average:50,stdev:0 GSM2317221_r1 2.67 3.38 0.09 109787218 138070043 104548358 132698759 125.76 126.93 1138376 907173 281.549 2792.909 111 3453 69.68 73.27 1296586 793239 1296586 793239 69.62 70.35 1296586 792554 1296586 761617 17558204 15.99 1.61 0 4.49 0 0.06 0 0.14 0 0.00 0 8.15 0 1138376 0 100 0 98.87 0 1.32 0 0.01 0 1.17 0 0.01 0 248.38 0 0.20 0 20017 0 1241919 0 55812 0 702 0 1686 0 0 0 101155 0 117 0 0 0 985 0 130371 0 785 0 132258 0 87.17 0 1082564 0 34058 133703 3.925744318515 1241919.0 1138376.0 20017.0 55812.0 702.0 1686.0 0.0 101155.0 1082564.0 91.7 1.6 4.5 0.1 0.1 0.0 8.1 87.2 50 50 50.00 24 62095950 26.7 21.8 21.8 29.7 0.0 36.5 23.2 smartseq 1441875 SRR4252002 SRP090061 SRS1699679 SRX2172003 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318221: SK_2_C12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318221 GSM2318221 SK_2_C12_smart-seq 182623100 1826231 2016-09-30 15:56:31 127156794 182623100 1826231 2 1826231 index:0,count:1826231,average:50,stdev:0|index:1,count:1826231,average:50,stdev:0 GSM2318221_r1 10.45 2.18 0.05 136665586 179984136 122296047 164390029 131.7 134.42 1514776 1363854 180.804 1087.961 78 8158 84.69 94.99 2013641 1282850 2013641 1282850 88.75 91.76 2013641 1344382 2013641 1239139 2486165 1.82 2.42 0 9.00 0 0.08 0 0.02 0 0.00 0 16.95 0 1514776 0 100 0 97.99 0 1.27 0 0.01 0 1.16 0 0.01 0 262.98 0 0.23 0 44273 0 1826231 0 164301 0 1450 0 414 0 0 0 309591 0 61 0 0 0 815 0 128144 0 784 0 129804 0 73.95 0 1350475 0 11284 130948 11.604750088621 1826231.0 1514776.0 44273.0 164301.0 1450.0 414.0 0.0 309591.0 1350475.0 82.9 2.4 9.0 0.1 0.0 0.0 17.0 73.9 50 50 50.00 38 91311550 25.9 22.4 22.3 29.5 0.0 36.9 21.1 smartseq 1441907 SRR4251003 SRP090061 SRS1698677 SRX2171004 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317222: 54Dn2_H02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317222 GSM2317222 54Dn2_H02_smart-seq 115424800 1154248 2016-09-30 15:56:31 51770262 115424800 1154248 2 1154248 index:0,count:1154248,average:50,stdev:0|index:1,count:1154248,average:50,stdev:0 GSM2317222_r1 1.43 3.49 0.14 99268025 126514397 94206693 121226156 127.45 128.68 1033561 819141 266.051 2541.861 110 3375 73.4 77.45 1173867 758669 1173867 758669 73.45 74.22 1173867 759139 1173867 727071 12008872 12.10 1.68 0 4.68 0 0.10 0 0.18 0 0.00 0 10.17 0 1033561 0 100 0 98.82 0 1.32 0 0.01 0 1.20 0 0.01 0 218.70 0 0.20 0 19388 0 1154248 0 53986 0 1192 0 2061 0 0 0 117434 0 103 0 0 0 1017 0 127613 0 690 0 129423 0 84.87 0 979575 0 31199 132355 4.242283406519 1154248.0 1033561.0 19388.0 53986.0 1192.0 2061.0 0.0 117434.0 979575.0 89.5 1.7 4.7 0.1 0.2 0.0 10.2 84.9 50 50 50.00 24 57712400 26.3 21.9 21.9 29.9 0.0 36.5 22.4 smartseq 1441908 SRR4252003 SRP090061 SRS1699681 SRX2172004 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318222: SK_2_D02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318222 GSM2318222 SK_2_D02_smart-seq 151558900 1515589 2016-09-30 15:56:31 107928478 151558900 1515589 2 1515589 index:0,count:1515589,average:50,stdev:0|index:1,count:1515589,average:50,stdev:0 GSM2318222_r1 7.56 2.98 0.08 129175420 172936042 120465459 162657581 133.88 135.02 1376093 1122875 235.392 2016.418 81 4807 77.32 83.1 1599322 1063981 1599322 1063981 78.54 79.47 1599322 1080735 1599322 1017523 12138102 9.40 2.07 0 6.31 0 0.07 0 0.06 0 0.00 0 9.07 0 1376093 0 100 0 98.45 0 1.34 0 0.01 0 1.19 0 0.01 0 272.81 0 0.28 0 31307 0 1515589 0 95655 0 1115 0 871 0 0 0 137510 0 159 0 0 0 1138 0 160807 0 944 0 163048 0 84.48 0 1280438 0 37290 164937 4.423089300080 1515589.0 1376093.0 31307.0 95655.0 1115.0 871.0 0.0 137510.0 1280438.0 90.8 2.1 6.3 0.1 0.1 0.0 9.1 84.5 50 50 50.00 38 75779450 27.0 22.1 22.3 28.5 0.0 37.1 23.8 smartseq 1441939 SRR4251004 SRP090061 SRS1698681 SRX2171005 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317223: 54Dn2_H03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317223 GSM2317223 54Dn2_H03_smart-seq 134489400 1344894 2016-09-30 15:56:31 61135217 134489400 1344894 2 1344894 index:0,count:1344894,average:50,stdev:0|index:1,count:1344894,average:50,stdev:0 GSM2317223_r1 2.78 3.48 0.04 117973235 156235397 111307384 148733263 132.43 133.62 1228223 957593 274.721 2736.785 111 3840 76.39 81.09 1406302 938263 1406302 938263 76.38 77.3 1406302 938151 1406302 894389 12274990 10.40 1.73 0 5.29 0 0.07 0 0.13 0 0.00 0 8.48 0 1228223 0 100 0 98.80 0 1.32 0 0.01 0 1.19 0 0.01 0 254.82 0 0.20 0 23231 0 1344894 0 71168 0 941 0 1733 0 0 0 113997 0 137 0 0 0 1198 0 159984 0 818 0 162137 0 86.03 0 1157055 0 39025 166032 4.254503523382 1344894.0 1228223.0 23231.0 71168.0 941.0 1733.0 0.0 113997.0 1157055.0 91.3 1.7 5.3 0.1 0.1 0.0 8.5 86.0 50 50 50.00 24 67244700 26.1 22.4 22.4 29.1 0.0 36.5 23.3 smartseq 1441940 SRR4252004 SRP090061 SRS1699682 SRX2172005 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318223: SK_2_D03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318223 GSM2318223 SK_2_D03_smart-seq 67942100 679421 2016-09-30 15:56:31 48856523 67942100 679421 2 679421 index:0,count:679421,average:50,stdev:0|index:1,count:679421,average:50,stdev:0 GSM2318223_r1 17.48 2.38 0.08 58341991 82057926 54282651 77958323 140.65 143.62 623021 542680 240.910 1892.993 106 2263 74.41 80.29 783008 463606 783008 463606 74.98 76.99 783008 467147 783008 444516 6324045 10.84 1.97 0 6.72 0 0.09 0 0.06 0 0.00 0 8.15 0 623021 0 100 0 98.39 0 1.28 0 0.01 0 1.19 0 0.01 0 163.06 0 0.32 0 13418 0 679421 0 45634 0 639 0 416 0 0 0 55345 0 30 0 0 0 381 0 46387 0 344 0 47142 0 84.98 0 577387 0 23126 46524 2.011761653550 679421.0 623021.0 13418.0 45634.0 639.0 416.0 0.0 55345.0 577387.0 91.7 2.0 6.7 0.1 0.1 0.0 8.1 85.0 50 50 50.00 38 33971050 27.2 21.8 22.1 28.8 0.0 37.0 23.6 smartseq 1441972 SRR4251005 SRP090061 SRS1698684 SRX2171006 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317224: 54Dn2_H04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317224 GSM2317224 54Dn2_H04_smart-seq 75043200 750432 2016-09-30 15:56:31 33604421 75043200 750432 2 750432 index:0,count:750432,average:50,stdev:0|index:1,count:750432,average:50,stdev:0 GSM2317224_r1 0.07 3.38 0.15 59461625 75419148 55910583 71742704 126.84 128.32 628289 517427 247.369 2268.849 100 2343 72.85 77.66 730891 457678 730891 457678 73.71 74.5 730891 463102 730891 439096 6897627 11.60 1.83 0 5.19 0 0.06 0 0.34 0 0.00 0 15.87 0 628289 0 100 0 98.76 0 1.33 0 0.01 0 1.20 0 0.01 0 142.19 0 0.19 0 13745 0 750432 0 38918 0 468 0 2570 0 0 0 119105 0 56 0 0 0 512 0 69378 0 412 0 70358 0 78.54 0 589371 0 20875 70865 3.394730538922 750432.0 628289.0 13745.0 38918.0 468.0 2570.0 0.0 119105.0 589371.0 83.7 1.8 5.2 0.1 0.3 0.0 15.9 78.5 50 50 50.00 24 37521600 25.7 21.7 21.4 31.1 0.0 36.3 20.9 smartseq 1441973 SRR4252005 SRP090061 SRS1699680 SRX2172006 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318224: SK_2_D04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318224 GSM2318224 SK_2_D04_smart-seq 177432900 1774329 2016-09-30 15:56:31 125240677 177432900 1774329 2 1774329 index:0,count:1774329,average:50,stdev:0|index:1,count:1774329,average:50,stdev:0 GSM2318224_r1 2.75 2.63 0.15 150585601 187899296 142603967 179346010 124.78 125.77 1619228 1413918 212.116 1720.644 100 6565 66.6 70.44 1855633 1078463 1855633 1078463 66.79 67.45 1855633 1081441 1855633 1032595 24743061 16.43 1.75 0 4.97 0 0.11 0 0.11 0 0.00 0 8.53 0 1619228 0 100 0 98.62 0 1.31 0 0.01 0 1.20 0 0.01 0 206.05 0 0.24 0 31020 0 1774329 0 88244 0 1866 0 1896 0 0 0 151339 0 132 0 0 0 1255 0 135218 0 1260 0 137865 0 86.29 0 1530984 0 29328 140308 4.784097108565 1774329.0 1619228.0 31020.0 88244.0 1866.0 1896.0 0.0 151339.0 1530984.0 91.3 1.7 5.0 0.1 0.1 0.0 8.5 86.3 50 50 50.00 38 88716450 27.5 21.6 21.8 29.2 0.0 37.2 23.9 smartseq 1442002 SRR4251006 SRP090061 SRS1698682 SRX2171007 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317225: 54Dn2_H05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317225 GSM2317225 54Dn2_H05_smart-seq 127293600 1272936 2016-09-30 15:56:31 56341692 127293600 1272936 2 1272936 index:0,count:1272936,average:50,stdev:0|index:1,count:1272936,average:50,stdev:0 GSM2317225_r1 3.45 3.26 0.1 108687284 142055983 103292529 136665146 130.7 132.31 1144164 930058 255.076 2289.137 100 3844 75.14 79.22 1324046 859707 1324046 859707 74.55 75.76 1324046 852982 1324046 822162 12779470 11.76 1.70 0 4.63 0 0.07 0 0.19 0 0.00 0 9.86 0 1144164 0 100 0 98.76 0 1.34 0 0.01 0 1.17 0 0.01 0 218.22 0 0.19 0 21680 0 1272936 0 58968 0 836 0 2460 0 0 0 125476 0 78 0 0 0 888 0 131836 0 741 0 133543 0 85.25 0 1085196 0 31884 133782 4.195897628905 1272936.0 1144164.0 21680.0 58968.0 836.0 2460.0 0.0 125476.0 1085196.0 89.9 1.7 4.6 0.1 0.2 0.0 9.9 85.3 50 50 50.00 24 63646800 26.5 21.8 21.9 29.8 0.0 36.5 23.0 smartseq 1442003 SRR4252006 SRP090061 SRS1699683 SRX2172007 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318225: SK_2_D05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318225 GSM2318225 SK_2_D05_smart-seq 213347700 2133477 2016-09-30 15:56:31 150507565 213347700 2133477 2 2133477 index:0,count:2133477,average:50,stdev:0|index:1,count:2133477,average:50,stdev:0 GSM2318225_r1 5.94 2.69 0.13 177729483 238569022 167306517 226523835 134.23 135.39 1912055 1597865 211.726 1899.880 78 7714 83.94 89.37 2204772 1604983 2204772 1604983 83.95 85.27 2204772 1605202 2204772 1531372 9471569 5.33 1.79 0 5.44 0 0.05 0 0.03 0 0.00 0 10.29 0 1912055 0 100 0 98.47 0 1.33 0 0.01 0 1.21 0 0.01 0 365.74 0 0.24 0 38176 0 2133477 0 116138 0 1116 0 726 0 0 0 219580 0 173 0 0 0 1943 0 214119 0 1171 0 217406 0 84.18 0 1795917 0 35441 221660 6.254338195875 2133477.0 1912055.0 38176.0 116138.0 1116.0 726.0 0.0 219580.0 1795917.0 89.6 1.8 5.4 0.1 0.0 0.0 10.3 84.2 50 50 50.00 38 106673850 27.0 22.0 22.3 28.6 0.0 37.2 23.8 smartseq 1442032 SRR4251007 SRP090061 SRS1698683 SRX2171008 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317226: 54Dn2_H06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317226 GSM2317226 54Dn2_H06_smart-seq 124670200 1246702 2016-09-30 15:56:31 55378052 124670200 1246702 2 1246702 index:0,count:1246702,average:50,stdev:0|index:1,count:1246702,average:50,stdev:0 GSM2317226_r1 5.7 3.27 0.11 104885687 136121504 99358745 130232373 129.78 131.07 1106208 910910 252.986 2489.612 112 3843 72.27 76.46 1273301 799447 1273301 799447 72.44 73.36 1273301 801287 1273301 766982 14273141 13.61 1.66 0 4.87 0 0.07 0 0.23 0 0.00 0 10.97 0 1106208 0 100 0 98.81 0 1.29 0 0.01 0 1.17 0 0.01 0 236.22 0 0.20 0 20645 0 1246702 0 60668 0 822 0 2897 0 0 0 136775 0 81 0 0 0 906 0 121093 0 707 0 122787 0 83.86 0 1045540 0 31277 123263 3.941010966525 1246702.0 1106208.0 20645.0 60668.0 822.0 2897.0 0.0 136775.0 1045540.0 88.7 1.7 4.9 0.1 0.2 0.0 11.0 83.9 50 50 50.00 24 62335100 26.4 21.8 21.7 30.0 0.0 36.5 22.7 smartseq 1442033 SRR4252007 SRP090061 SRS1699686 SRX2172008 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318226: SK_2_D06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318226 GSM2318226 SK_2_D06_smart-seq 12484700 124847 2016-09-30 15:56:31 8937053 12484700 124847 2 124847 index:0,count:124847,average:50,stdev:0|index:1,count:124847,average:50,stdev:0 GSM2318226_r1 5.35 2.49 0.15 8120326 9678037 7161114 8908619 119.18 124.4 92274 85448 177.964 3298.297 54 615 52.32 59.76 136854 48274 136854 48274 54.24 57.83 136854 50052 136854 46719 1719645 21.18 3.72 0 9.20 0 0.44 0 0.40 0 0.00 0 25.25 0 92274 0 100 0 96.83 0 1.40 0 0.02 0 1.17 0 0.01 0 32.10 0 0.53 0 4644 0 124847 0 11492 0 550 0 497 0 0 0 31526 0 2 0 0 0 32 0 3364 0 69 0 3467 0 64.70 0 80782 0 2030 3302 1.626600985222 124847.0 92274.0 4644.0 11492.0 550.0 497.0 0.0 31526.0 80782.0 73.9 3.7 9.2 0.4 0.4 0.0 25.3 64.7 50 50 50.00 38 6242350 25.7 21.4 21.2 31.7 0.0 36.5 18.9 smartseq 1442065 SRR4251008 SRP090061 SRS1698687 SRX2171009 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317227: 54Dn2_H07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317227 GSM2317227 54Dn2_H07_smart-seq 168391800 1683918 2016-09-30 15:56:31 74841131 168391800 1683918 2 1683918 index:0,count:1683918,average:50,stdev:0|index:1,count:1683918,average:50,stdev:0 GSM2317227_r1 5.27 3.91 0.06 147435789 197148587 138157211 187296513 133.72 135.57 1545115 1223800 255.852 2667.060 100 5363 79.66 85.18 1836276 1230775 1836276 1230775 79.96 81.48 1836276 1235408 1836276 1177295 11640349 7.90 1.74 0 5.95 0 0.05 0 0.14 0 0.00 0 8.05 0 1545115 0 100 0 98.78 0 1.29 0 0.01 0 1.18 0 0.01 0 242.48 0 0.19 0 29340 0 1683918 0 100187 0 896 0 2424 0 0 0 135483 0 129 0 0 0 1412 0 200579 0 1009 0 203129 0 85.81 0 1444928 0 42788 206581 4.828012526877 1683918.0 1545115.0 29340.0 100187.0 896.0 2424.0 0.0 135483.0 1444928.0 91.8 1.7 5.9 0.1 0.1 0.0 8.0 85.8 50 50 50.00 24 84195900 26.5 22.2 22.3 29.0 0.0 36.6 24.3 smartseq 1442066 SRR4252008 SRP090061 SRS1699685 SRX2172009 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318227: SK_2_D07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318227 GSM2318227 SK_2_D07_smart-seq 181977900 1819779 2016-09-30 15:56:31 128845873 181977900 1819779 2 1819779 index:0,count:1819779,average:50,stdev:0|index:1,count:1819779,average:50,stdev:0 GSM2318227_r1 6.04 3.45 0.15 155850114 200672970 148076110 192384443 128.76 129.92 1670546 1421744 215.524 2100.197 81 6517 71.73 75.63 1896908 1198201 1896908 1198201 71.62 72.43 1896908 1196415 1896908 1147411 20065913 12.88 1.77 0 4.74 0 0.08 0 0.10 0 0.00 0 8.02 0 1670546 0 100 0 98.61 0 1.31 0 0.01 0 1.21 0 0.01 0 211.33 0 0.26 0 32205 0 1819779 0 86298 0 1433 0 1778 0 0 0 146022 0 87 0 0 0 1421 0 160989 0 1175 0 163672 0 87.06 0 1584248 0 36967 165859 4.486677306787 1819779.0 1670546.0 32205.0 86298.0 1433.0 1778.0 0.0 146022.0 1584248.0 91.8 1.8 4.7 0.1 0.1 0.0 8.0 87.1 50 50 50.00 38 90988950 27.4 21.8 22.0 28.8 0.0 37.2 24.1 smartseq 1442097 SRR4251009 SRP090061 SRS1698685 SRX2171010 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317228: 54Dn2_H08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317228 GSM2317228 54Dn2_H08_smart-seq 122113400 1221134 2016-09-30 15:56:31 54006515 122113400 1221134 2 1221134 index:0,count:1221134,average:50,stdev:0|index:1,count:1221134,average:50,stdev:0 GSM2317228_r1 3.64 0.75 0.03 101671276 134957355 95378677 127709266 132.74 133.9 1079205 869224 239.186 2145.692 100 4027 77.21 82.5 1261863 833236 1261863 833236 77.36 78.54 1261863 834893 1261863 793220 9723343 9.56 1.92 0 5.67 0 0.12 0 0.25 0 0.00 0 11.25 0 1079205 0 100 0 98.62 0 1.32 0 0.01 0 1.15 0 0.01 0 244.23 0 0.20 0 23457 0 1221134 0 69258 0 1491 0 3034 0 0 0 137404 0 175 0 0 0 1012 0 142627 0 764 0 144578 0 82.71 0 1009947 0 27736 147164 5.305884049611 1221134.0 1079205.0 23457.0 69258.0 1491.0 3034.0 0.0 137404.0 1009947.0 88.4 1.9 5.7 0.1 0.2 0.0 11.3 82.7 50 50 50.00 24 61056700 26.0 22.5 22.4 29.1 0.0 36.6 23.6 smartseq 1442098 SRR4252009 SRP090061 SRS1699687 SRX2172010 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318228: SK_2_D08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318228 GSM2318228 SK_2_D08_smart-seq 82522200 825222 2016-09-30 15:56:31 59043954 82522200 825222 2 825222 index:0,count:825222,average:50,stdev:0|index:1,count:825222,average:50,stdev:0 GSM2318228_r1 16.4 2.4 0.09 70663070 98516716 65510689 93528337 139.42 142.77 756022 658739 236.694 1900.443 99 2750 73.77 79.88 970832 557743 970832 557743 74.41 76.7 970832 562534 970832 535522 7923822 11.21 1.98 0 7.01 0 0.10 0 0.07 0 0.00 0 8.22 0 756022 0 100 0 98.40 0 1.30 0 0.01 0 1.20 0 0.01 0 174.75 0 0.31 0 16366 0 825222 0 57833 0 784 0 554 0 0 0 67862 0 34 0 0 0 468 0 57357 0 448 0 58307 0 84.61 0 698189 0 26045 57181 2.195469379919 825222.0 756022.0 16366.0 57833.0 784.0 554.0 0.0 67862.0 698189.0 91.6 2.0 7.0 0.1 0.1 0.0 8.2 84.6 50 50 50.00 38 41261100 27.2 21.9 22.2 28.7 0.0 37.1 23.7 smartseq 1442321 SRR4251010 SRP090061 SRS1698686 SRX2171011 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317229: 54Dn2_H09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317229 GSM2317229 54Dn2_H09_smart-seq 106209600 1062096 2016-09-30 15:56:31 46897309 106209600 1062096 2 1062096 index:0,count:1062096,average:50,stdev:0|index:1,count:1062096,average:50,stdev:0 GSM2317229_r1 4.36 2.98 0.1 90553148 122432177 85583947 116794746 135.2 136.47 959633 725326 251.001 2566.214 111 3183 84.26 89.37 1098169 808605 1098169 808605 84.13 85.24 1098169 807349 1098169 771249 5198651 5.74 1.99 0 5.16 0 0.07 0 0.16 0 0.00 0 9.42 0 959633 0 100 0 98.56 0 1.28 0 0.01 0 1.16 0 0.01 0 224.91 0 0.19 0 21102 0 1062096 0 54799 0 706 0 1674 0 0 0 100083 0 61 0 0 0 977 0 149634 0 650 0 151322 0 85.19 0 904834 0 37838 152241 4.023494899308 1062096.0 959633.0 21102.0 54799.0 706.0 1674.0 0.0 100083.0 904834.0 90.4 2.0 5.2 0.1 0.2 0.0 9.4 85.2 50 50 50.00 24 53104800 26.1 22.4 22.4 29.0 0.0 36.6 23.8 smartseq 1442322 SRR4252010 SRP090061 SRS1699684 SRX2172011 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318229: SK_2_D09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318229 GSM2318229 SK_2_D09_smart-seq 148452400 1484524 2016-09-30 15:56:31 104586138 148452400 1484524 2 1484524 index:0,count:1484524,average:50,stdev:0|index:1,count:1484524,average:50,stdev:0 GSM2318229_r1 1.85 3.12 0.09 125737583 160117349 117383421 150984689 127.34 128.63 1350087 1103141 223.515 1959.399 78 5185 74.1 79.54 1589967 1000473 1589967 1000473 74.76 75.79 1589967 1009392 1589967 953293 14620004 11.63 2.04 0 6.22 0 0.10 0 0.11 0 0.00 0 8.85 0 1350087 0 100 0 98.45 0 1.39 0 0.01 0 1.21 0 0.01 0 242.92 0 0.25 0 30355 0 1484524 0 92317 0 1468 0 1632 0 0 0 131337 0 167 0 0 0 1202 0 164556 0 997 0 166922 0 84.73 0 1257770 0 39037 168934 4.327535415119 1484524.0 1350087.0 30355.0 92317.0 1468.0 1632.0 0.0 131337.0 1257770.0 90.9 2.0 6.2 0.1 0.1 0.0 8.8 84.7 50 50 50.00 38 74226200 26.9 22.3 22.5 28.4 0.0 37.3 24.2 smartseq 1442352 SRR4251011 SRP090061 SRS1698690 SRX2171012 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317230: 54Dn2_H10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317230 GSM2317230 54Dn2_H10_smart-seq 131307800 1313078 2016-09-30 15:56:31 58677415 131307800 1313078 2 1313078 index:0,count:1313078,average:50,stdev:0|index:1,count:1313078,average:50,stdev:0 GSM2317230_r1 4.81 3.54 0.11 108493468 144294486 101958617 137198150 133.0 134.56 1156888 930120 238.829 2393.072 91 4393 78.4 83.65 1354489 906983 1354489 906983 78.55 79.81 1354489 908688 1354489 865375 10225349 9.42 1.92 0 5.53 0 0.06 0 0.22 0 0.00 0 11.61 0 1156888 0 100 0 98.62 0 1.28 0 0.01 0 1.19 0 0.01 0 262.62 0 0.19 0 25238 0 1313078 0 72606 0 838 0 2877 0 0 0 152475 0 105 0 0 0 1006 0 151086 0 778 0 152975 0 82.58 0 1084282 0 33743 153643 4.553329579468 1313078.0 1156888.0 25238.0 72606.0 838.0 2877.0 0.0 152475.0 1084282.0 88.1 1.9 5.5 0.1 0.2 0.0 11.6 82.6 50 50 50.00 24 65653900 26.0 22.3 22.2 29.4 0.0 36.5 23.0 smartseq 1442353 SRR4252011 SRP090061 SRS1699688 SRX2172012 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318230: SK_2_D10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318230 GSM2318230 SK_2_D10_smart-seq 215062000 2150620 2016-09-30 15:56:31 152096068 215062000 2150620 2 2150620 index:0,count:2150620,average:50,stdev:0|index:1,count:2150620,average:50,stdev:0 GSM2318230_r1 5.09 3.19 0.11 176577057 226339845 164770191 213096874 128.18 129.33 1918285 1647156 199.534 1615.423 81 8675 72.65 78.04 2263432 1393600 2263432 1393600 74.05 74.76 2263432 1420506 2263432 1335099 21172296 11.99 2.02 0 6.16 0 0.11 0 0.09 0 0.00 0 10.61 0 1918285 0 100 0 98.43 0 1.35 0 0.01 0 1.18 0 0.01 0 286.75 0 0.25 0 43508 0 2150620 0 132518 0 2296 0 1900 0 0 0 228139 0 207 0 0 0 1464 0 200375 0 1458 0 203504 0 83.03 0 1785767 0 32683 206814 6.327876877888 2150620.0 1918285.0 43508.0 132518.0 2296.0 1900.0 0.0 228139.0 1785767.0 89.2 2.0 6.2 0.1 0.1 0.0 10.6 83.0 50 50 50.00 38 107531000 26.9 22.1 22.3 28.7 0.0 37.2 23.6 smartseq 1442384 SRR4251012 SRP090061 SRS1698688 SRX2171013 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317231: 54Dn2_H11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317231 GSM2317231 54Dn2_H11_smart-seq 124721600 1247216 2016-09-30 15:56:31 55077101 124721600 1247216 2 1247216 index:0,count:1247216,average:50,stdev:0|index:1,count:1247216,average:50,stdev:0 GSM2317231_r1 2.58 3.8 0.1 97982488 126490762 92421594 120736989 129.1 130.64 1050939 863760 229.948 2121.747 54 4517 74.52 79.24 1217388 783139 1217388 783139 74.9 75.98 1217388 787103 1217388 750983 11104929 11.33 1.90 0 5.02 0 0.09 0 0.36 0 0.00 0 15.29 0 1050939 0 100 0 98.59 0 1.28 0 0.01 0 1.18 0 0.01 0 236.31 0 0.19 0 23658 0 1247216 0 62571 0 1093 0 4453 0 0 0 190731 0 92 0 0 0 1057 0 128063 0 747 0 129959 0 79.25 0 988368 0 26293 131026 4.983303540866 1247216.0 1050939.0 23658.0 62571.0 1093.0 4453.0 0.0 190731.0 988368.0 84.3 1.9 5.0 0.1 0.4 0.0 15.3 79.2 50 50 50.00 24 62360800 26.0 21.9 21.7 30.4 0.0 36.5 22.1 smartseq 1442385 SRR4252012 SRP090061 SRS1699690 SRX2172013 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318231: SK_2_D11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318231 GSM2318231 SK_2_D11_smart-seq 174143600 1741436 2016-09-30 15:56:31 123063491 174143600 1741436 2 1741436 index:0,count:1741436,average:50,stdev:0|index:1,count:1741436,average:50,stdev:0 GSM2318231_r1 3.64 3.36 0.22 149025388 190389408 141082513 181243802 127.76 128.47 1600692 1364093 217.143 1991.380 81 6335 71.71 75.89 1823560 1147881 1823560 1147881 71.74 72.38 1823560 1148263 1823560 1094770 20739670 13.92 1.80 0 5.06 0 0.08 0 0.08 0 0.00 0 7.92 0 1600692 0 100 0 98.59 0 1.35 0 0.01 0 1.20 0 0.01 0 216.18 0 0.25 0 31414 0 1741436 0 88102 0 1432 0 1415 0 0 0 137897 0 156 0 0 0 1208 0 155050 0 1277 0 157691 0 86.86 0 1512590 0 34385 160486 4.667325868838 1741436.0 1600692.0 31414.0 88102.0 1432.0 1415.0 0.0 137897.0 1512590.0 91.9 1.8 5.1 0.1 0.1 0.0 7.9 86.9 50 50 50.00 38 87071800 27.4 21.7 21.9 29.0 0.0 37.2 24.0 smartseq 1442416 SRR4251013 SRP090061 SRS1698689 SRX2171014 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317232: 26Dp4_A01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317232 GSM2317232 26Dp4_A01_smart-seq 212781100 2127811 2016-09-30 15:56:31 148648929 212781100 2127811 2 2127811 index:0,count:2127811,average:50,stdev:0|index:1,count:2127811,average:50,stdev:0 GSM2317232_r1 3.32 3.08 0.13 184150997 226953941 175829593 218554769 123.24 124.3 1950551 1664692 251.861 2304.043 110 6355 63.71 66.82 2211876 1242613 2211876 1242613 63.51 64.14 2211876 1238833 2211876 1192851 37070543 20.13 1.80 0 4.27 0 0.10 0 0.13 0 0.00 0 8.10 0 1950551 0 100 0 98.72 0 1.36 0 0.01 0 1.21 0 0.01 0 306.40 0 0.28 0 38246 0 2127811 0 90866 0 2095 0 2711 0 0 0 172454 0 96 0 0 0 1338 0 159336 0 1337 0 162107 0 87.40 0 1859685 0 29939 164934 5.509001636661 2127811.0 1950551.0 38246.0 90866.0 2095.0 2711.0 0.0 172454.0 1859685.0 91.7 1.8 4.3 0.1 0.1 0.0 8.1 87.4 50 50 50.00 38 106390550 27.8 21.3 21.5 29.4 0.0 37.2 23.6 smartseq 1442417 SRR4252013 SRP090061 SRS1699691 SRX2172014 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318232: SK_2_D12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318232 GSM2318232 SK_2_D12_smart-seq 114924500 1149245 2016-09-30 15:56:31 80843424 114924500 1149245 2 1149245 index:0,count:1149245,average:50,stdev:0|index:1,count:1149245,average:50,stdev:0 GSM2318232_r1 4.18 1.1 0.09 75817794 82706218 71060032 79513575 109.09 111.9 867875 836752 145.703 793.615 57 6169 76.54 81.83 1143356 664282 1143356 664282 76.97 80.41 1143356 667993 1143356 652826 5301537 6.99 2.01 0 4.88 0 0.10 0 0.07 0 0.00 0 24.31 0 867875 0 100 0 97.58 0 1.35 0 0.01 0 1.15 0 0.01 0 217.75 0 0.23 0 23114 0 1149245 0 56046 0 1206 0 838 0 0 0 279326 0 30 0 0 0 205 0 26585 0 367 0 27187 0 70.64 0 811829 0 3150 26662 8.464126984127 1149245.0 867875.0 23114.0 56046.0 1206.0 838.0 0.0 279326.0 811829.0 75.5 2.0 4.9 0.1 0.1 0.0 24.3 70.6 50 50 50.00 38 57462250 25.9 21.6 21.5 31.0 0.0 36.7 19.8 smartseq 1442448 SRR4251014 SRP090061 SRS1698692 SRX2171015 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317233: 26Dp4_A02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317233 GSM2317233 26Dp4_A02_smart-seq 214279800 2142798 2016-09-30 15:56:31 150059694 214279800 2142798 2 2142798 index:0,count:2142798,average:50,stdev:0|index:1,count:2142798,average:50,stdev:0 GSM2317233_r1 3.77 3.17 0.19 186319842 235376228 175981643 224604766 126.33 127.63 1969970 1645180 257.241 2444.828 81 6196 66.58 70.61 2293032 1311591 2293032 1311591 66.9 67.66 2293032 1317983 2293032 1256710 31625804 16.97 1.88 0 5.25 0 0.10 0 0.11 0 0.00 0 7.85 0 1969970 0 100 0 98.69 0 1.33 0 0.01 0 1.23 0 0.01 0 285.71 0 0.29 0 40337 0 2142798 0 112575 0 2135 0 2423 0 0 0 168270 0 144 0 0 0 1774 0 182548 0 1523 0 185989 0 86.68 0 1857395 0 37409 190495 5.092223796413 2142798.0 1969970.0 40337.0 112575.0 2135.0 2423.0 0.0 168270.0 1857395.0 91.9 1.9 5.3 0.1 0.1 0.0 7.9 86.7 50 50 50.00 38 107139900 27.2 21.9 22.1 28.7 0.0 37.2 23.7 smartseq 1442449 SRR4252014 SRP090061 SRS1699692 SRX2172015 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318233: SK_2_E01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318233 GSM2318233 SK_2_E01_smart-seq 305270600 3052706 2016-09-30 15:56:31 212686307 305270600 3052706 2 3052706 index:0,count:3052706,average:50,stdev:0|index:1,count:3052706,average:50,stdev:0 GSM2318233_r1 3.17 2.86 0.18 252746360 320455211 240197371 306339427 126.79 127.54 2758240 2422671 198.335 1610.936 83 12367 69.78 73.57 3103731 1924711 3103731 1924711 70.14 70.65 3103731 1934596 3103731 1848461 37008124 14.64 1.97 0 4.65 0 0.10 0 0.12 0 0.00 0 9.43 0 2758240 0 100 0 98.45 0 1.36 0 0.01 0 1.19 0 0.01 0 249.77 0 0.22 0 60094 0 3052706 0 142001 0 2990 0 3598 0 0 0 287878 0 133 0 0 0 1865 0 241182 0 2232 0 245412 0 85.70 0 2616239 0 31937 249560 7.814134076463 3052706.0 2758240.0 60094.0 142001.0 2990.0 3598.0 0.0 287878.0 2616239.0 90.4 2.0 4.7 0.1 0.1 0.0 9.4 85.7 50 50 50.00 38 152635300 27.2 21.7 21.8 29.2 0.0 37.2 23.5 smartseq 1442483 SRR4251015 SRP090061 SRS1698691 SRX2171016 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317234: 26Dp4_A03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317234 GSM2317234 26Dp4_A03_smart-seq 242940500 2429405 2016-09-30 15:56:31 170395885 242940500 2429405 2 2429405 index:0,count:2429405,average:50,stdev:0|index:1,count:2429405,average:50,stdev:0 GSM2317234_r1 1.71 3.14 0.06 204042711 265091132 188540900 250140069 129.92 132.67 2176001 1748275 231.076 2620.483 100 7979 89.3 96.82 2772615 1943085 2772615 1943085 89.3 92.41 2772615 1943274 2772615 1854487 2296456 1.13 1.83 0 6.96 0 0.05 0 0.02 0 0.00 0 10.35 0 2176001 0 100 0 98.40 0 1.37 0 0.01 0 1.21 0 0.01 0 282.12 0 0.27 0 44481 0 2429405 0 169178 0 1260 0 587 0 0 0 251557 0 207 0 0 0 2124 0 279136 0 1192 0 282659 0 82.61 0 2006823 0 23846 287706 12.065168162375 2429405.0 2176001.0 44481.0 169178.0 1260.0 587.0 0.0 251557.0 2006823.0 89.6 1.8 7.0 0.1 0.0 0.0 10.4 82.6 50 50 50.00 38 121470250 26.2 22.9 23.1 27.8 0.0 37.2 23.1 smartseq 1442484 SRR4252015 SRP090061 SRS1699689 SRX2172016 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318234: SK_2_E02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318234 GSM2318234 SK_2_E02_smart-seq 227540800 2275408 2016-09-30 15:56:31 159368482 227540800 2275408 2 2275408 index:0,count:2275408,average:50,stdev:0|index:1,count:2275408,average:50,stdev:0 GSM2318234_r1 1.52 3.24 0.09 192246456 250014711 181608704 238166222 130.05 131.14 2072237 1710786 218.245 2046.202 81 8001 76.41 81.04 2371650 1583385 2371650 1583385 76.76 77.65 2371650 1590622 2371650 1517284 21633366 11.25 1.95 0 5.20 0 0.09 0 0.09 0 0.00 0 8.76 0 2072237 0 100 0 98.43 0 1.37 0 0.01 0 1.21 0 0.01 0 240.93 0 0.23 0 44378 0 2275408 0 118295 0 1968 0 1964 0 0 0 199239 0 204 0 0 0 1875 0 242059 0 1551 0 245689 0 85.87 0 1953942 0 38862 247205 6.361098245072 2275408.0 2072237.0 44378.0 118295.0 1968.0 1964.0 0.0 199239.0 1953942.0 91.1 2.0 5.2 0.1 0.1 0.0 8.8 85.9 50 50 50.00 38 113770400 27.0 22.1 22.2 28.7 0.0 37.2 23.8 smartseq 1442512 SRR4252016 SRP090061 SRS1699693 SRX2172017 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318235: SK_2_E03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318235 GSM2318235 SK_2_E03_smart-seq 274105600 2741056 2016-09-30 15:56:31 193146734 274105600 2741056 2 2741056 index:0,count:2741056,average:50,stdev:0|index:1,count:2741056,average:50,stdev:0 GSM2318235_r1 6.67 2.61 0.12 233501847 308765869 217290627 290082514 132.23 133.5 2511754 2056886 225.057 1888.361 83 9467 76.77 82.73 2948552 1928194 2948552 1928194 78.02 79.03 2948552 1959728 2948552 1842006 22383390 9.59 2.21 0 6.60 0 0.07 0 0.07 0 0.00 0 8.23 0 2511754 0 100 0 98.40 0 1.34 0 0.01 0 1.19 0 0.01 0 394.71 0 0.25 0 60594 0 2741056 0 180973 0 1906 0 1849 0 0 0 225547 0 293 0 0 0 1966 0 301445 0 1703 0 305407 0 85.03 0 2330781 0 43709 309004 7.069573771992 2741056.0 2511754.0 60594.0 180973.0 1906.0 1849.0 0.0 225547.0 2330781.0 91.6 2.2 6.6 0.1 0.1 0.0 8.2 85.0 50 50 50.00 38 137052800 27.0 22.2 22.3 28.5 0.0 37.1 24.0 smartseq 1442544 SRR4251017 SRP090061 SRS1698693 SRX2171018 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317236: 26Dp4_A05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317236 GSM2317236 26Dp4_A05_smart-seq 213712100 2137121 2016-09-30 15:56:31 148732596 213712100 2137121 2 2137121 index:0,count:2137121,average:50,stdev:0|index:1,count:2137121,average:50,stdev:0 GSM2317236_r1 2.4 3.08 0.13 187033097 234600455 177282411 224307378 125.43 126.53 1963609 1626558 254.938 2290.017 110 6206 69.11 73.02 2258970 1357138 2258970 1357138 69.14 69.79 2258970 1357633 2258970 1297104 30293267 16.20 1.82 0 4.91 0 0.08 0 0.11 0 0.00 0 7.93 0 1963609 0 100 0 98.71 0 1.41 0 0.01 0 1.22 0 0.01 0 240.43 0 0.26 0 39000 0 2137121 0 104972 0 1643 0 2426 0 0 0 169443 0 93 0 0 0 1692 0 195452 0 1482 0 198719 0 86.97 0 1858637 0 30563 203721 6.665608742597 2137121.0 1963609.0 39000.0 104972.0 1643.0 2426.0 0.0 169443.0 1858637.0 91.9 1.8 4.9 0.1 0.1 0.0 7.9 87.0 50 50 50.00 38 106856050 27.2 21.9 22.1 28.8 0.0 37.2 23.5 smartseq 1442545 SRR4252017 SRP090061 SRS1699695 SRX2172018 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318236: SK_2_E04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318236 GSM2318236 SK_2_E04_smart-seq 242338300 2423383 2016-09-30 15:56:31 170049105 242338300 2423383 2 2423383 index:0,count:2423383,average:50,stdev:0|index:1,count:2423383,average:50,stdev:0 GSM2318236_r1 4.29 3.07 0.16 204122360 257430814 189917484 242758648 126.12 127.82 2210508 1920220 207.341 1585.304 83 9247 68.09 73.37 2675158 1505173 2675158 1505173 69.41 70.36 2675158 1534399 2675158 1443551 29827230 14.61 2.07 0 6.56 0 0.11 0 0.09 0 0.00 0 8.58 0 2210508 0 100 0 98.49 0 1.36 0 0.01 0 1.20 0 0.01 0 300.83 0 0.23 0 50196 0 2423383 0 158958 0 2648 0 2224 0 0 0 208003 0 179 0 0 0 1578 0 199988 0 1371 0 203116 0 84.66 0 2051550 0 32500 205424 6.320738461538 2423383.0 2210508.0 50196.0 158958.0 2648.0 2224.0 0.0 208003.0 2051550.0 91.2 2.1 6.6 0.1 0.1 0.0 8.6 84.7 50 50 50.00 38 121169150 27.1 21.9 22.0 29.1 0.0 37.1 23.4 smartseq 1442960 SRR4252024 SRP090061 SRS1699701 SRX2172025 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318243: SK_2_E11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318243 GSM2318243 SK_2_E11_smart-seq 281805900 2818059 2016-09-30 15:56:31 196276741 281805900 2818059 2 2818059 index:0,count:2818059,average:50,stdev:0|index:1,count:2818059,average:50,stdev:0 GSM2318243_r1 5.62 2.94 0.12 237026381 310932440 218723721 292607191 131.18 133.78 2577771 2173980 202.887 1786.484 83 11391 74.33 80.8 3243267 1916147 3243267 1916147 75.63 77.39 3243267 1949461 3243267 1835348 22706675 9.58 2.16 0 7.32 0 0.10 0 0.08 0 0.00 0 8.34 0 2577771 0 100 0 98.36 0 1.34 0 0.01 0 1.21 0 0.01 0 253.63 0 0.23 0 60742 0 2818059 0 206251 0 2862 0 2289 0 0 0 235137 0 199 0 0 0 2217 0 292502 0 2115 0 297033 0 84.15 0 2371520 0 48647 298965 6.145599934220 2818059.0 2577771.0 60742.0 206251.0 2862.0 2289.0 0.0 235137.0 2371520.0 91.5 2.2 7.3 0.1 0.1 0.0 8.3 84.2 50 50 50.00 38 140902950 26.8 22.2 22.3 28.7 0.0 37.2 23.7 smartseq 1442992 SRR4251025 SRP090061 SRS1698702 SRX2171026 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317244: 26Dp4_B02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317244 GSM2317244 26Dp4_B02_smart-seq 246559500 2465595 2016-09-30 15:56:31 171287598 246559500 2465595 2 2465595 index:0,count:2465595,average:50,stdev:0|index:1,count:2465595,average:50,stdev:0 GSM2317244_r1 2.81 3.12 0.17 216566960 274539787 205140151 262368329 126.77 127.9 2285777 1896460 254.003 2544.465 100 7335 69.79 73.79 2622536 1595156 2622536 1595156 69.84 70.64 2622536 1596294 2622536 1527213 31906145 14.73 1.80 0 5.02 0 0.08 0 0.11 0 0.00 0 7.10 0 2285777 0 100 0 98.72 0 1.36 0 0.01 0 1.20 0 0.01 0 306.07 0 0.27 0 44295 0 2465595 0 123881 0 2001 0 2664 0 0 0 175153 0 181 0 0 0 1837 0 224942 0 1897 0 228857 0 87.68 0 2161896 0 35930 235709 6.560228221542 2465595.0 2285777.0 44295.0 123881.0 2001.0 2664.0 0.0 175153.0 2161896.0 92.7 1.8 5.0 0.1 0.1 0.0 7.1 87.7 50 50 50.00 38 123279750 27.2 22.0 22.2 28.6 0.0 37.2 23.8 smartseq 1442993 SRR4252025 SRP090061 SRS1699703 SRX2172026 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318244: SK_2_E12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318244 GSM2318244 SK_2_E12_smart-seq 308498800 3084988 2016-09-30 15:56:31 213529799 308498800 3084988 2 3084988 index:0,count:3084988,average:50,stdev:0|index:1,count:3084988,average:50,stdev:0 GSM2318244_r1 5.25 2.61 0.17 253045521 329407673 237487452 312199802 130.18 131.46 2790858 2469969 180.382 1437.179 78 14475 73.58 78.61 3291101 2053647 3291101 2053647 73.52 74.59 3291101 2051825 3291101 1948582 31339215 12.38 2.06 0 5.78 0 0.08 0 0.10 0 0.00 0 9.35 0 2790858 0 100 0 98.38 0 1.30 0 0.01 0 1.19 0 0.01 0 370.20 0 0.22 0 63401 0 3084988 0 178367 0 2456 0 3165 0 0 0 288509 0 180 0 0 0 2360 0 266619 0 2135 0 271294 0 84.68 0 2612491 0 31790 275531 8.667222396980 3084988.0 2790858.0 63401.0 178367.0 2456.0 3165.0 0.0 288509.0 2612491.0 90.5 2.1 5.8 0.1 0.1 0.0 9.4 84.7 50 50 50.00 38 154249400 26.9 22.0 22.1 29.0 0.0 37.3 23.3 smartseq 1443024 SRR4252026 SRP090061 SRS1699702 SRX2172027 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318245: SK_2_F01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318245 GSM2318245 SK_2_F01_smart-seq 324076800 3240768 2016-09-30 15:56:31 224494806 324076800 3240768 2 3240768 index:0,count:3240768,average:50,stdev:0|index:1,count:3240768,average:50,stdev:0 GSM2318245_r1 2.76 2.98 0.19 273096959 337954624 257817941 322693739 123.75 125.16 2970388 2613991 193.213 1712.961 81 13792 65.1 69.09 3483964 1933835 3483964 1933835 65.11 66.04 3483964 1934120 3483964 1848262 48024456 17.59 1.86 0 5.29 0 0.11 0 0.11 0 0.00 0 8.12 0 2970388 0 100 0 98.54 0 1.35 0 0.01 0 1.20 0 0.01 0 402.30 0 0.22 0 60291 0 3240768 0 171520 0 3450 0 3694 0 0 0 263236 0 279 0 0 0 2057 0 262164 0 2256 0 266756 0 86.36 0 2798868 0 39089 271621 6.948783545243 3240768.0 2970388.0 60291.0 171520.0 3450.0 3694.0 0.0 263236.0 2798868.0 91.7 1.9 5.3 0.1 0.1 0.0 8.1 86.4 50 50 50.00 38 162038400 27.6 21.4 21.5 29.5 0.0 37.3 23.6 smartseq 1443056 SRR4252027 SRP090061 SRS1699704 SRX2172028 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318246: SK_2_F02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318246 GSM2318246 SK_2_F02_smart-seq 65000300 650003 2016-09-30 15:56:31 45359559 65000300 650003 2 650003 index:0,count:650003,average:50,stdev:0|index:1,count:650003,average:50,stdev:0 GSM2318246_r1 6.49 1.15 0.03 37406514 44274418 33257543 40416110 118.36 121.52 432186 411159 150.323 811.859 51 3394 86.33 97.46 622406 373088 622406 373088 90.85 95.62 622406 392659 622406 366048 269547 0.72 2.31 0 7.59 0 0.11 0 0.01 0 0.00 0 33.40 0 432186 0 100 0 97.40 0 1.26 0 0.01 0 1.19 0 0.01 0 123.16 0 0.21 0 14984 0 650003 0 49359 0 713 0 33 0 0 0 217071 0 23 0 0 0 125 0 22019 0 95 0 22262 0 58.90 0 382827 0 2670 22574 8.454681647940 650003.0 432186.0 14984.0 49359.0 713.0 33.0 0.0 217071.0 382827.0 66.5 2.3 7.6 0.1 0.0 0.0 33.4 58.9 50 50 50.00 38 32500150 24.7 21.8 21.2 32.3 0.0 36.2 17.8 smartseq 1443888 SRR4251041 SRP090061 SRS1698717 SRX2171042 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317260: 26Dp4_C07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317260 GSM2317260 26Dp4_C07_smart-seq 223076400 2230764 2016-09-30 15:56:31 154728945 223076400 2230764 2 2230764 index:0,count:2230764,average:50,stdev:0|index:1,count:2230764,average:50,stdev:0 GSM2317260_r1 3.42 3.2 0.26 191753899 237262516 182581025 228338469 123.73 125.06 2035722 1716855 253.030 2607.573 80 6504 64.86 68.23 2339803 1320375 2339803 1320375 64.94 65.7 2339803 1321983 2339803 1271279 36717551 19.15 1.83 0 4.51 0 0.10 0 0.15 0 0.00 0 8.50 0 2035722 0 100 0 98.69 0 1.38 0 0.01 0 1.20 0 0.01 0 308.88 0 0.27 0 40783 0 2230764 0 100653 0 2133 0 3246 0 0 0 189663 0 156 0 0 0 1464 0 178791 0 1699 0 182110 0 86.74 0 1935069 0 33445 184666 5.521483031843 2230764.0 2035722.0 40783.0 100653.0 2133.0 3246.0 0.0 189663.0 1935069.0 91.3 1.8 4.5 0.1 0.1 0.0 8.5 86.7 50 50 50.00 38 111538200 27.5 21.7 21.7 29.1 0.0 37.2 23.5 smartseq 1443889 SRR4252041 SRP090061 SRS1699719 SRX2172042 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318260: SK_2_G04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318260 GSM2318260 SK_2_G04_smart-seq 210295300 2102953 2016-09-30 15:56:31 149638832 210295300 2102953 2 2102953 index:0,count:2102953,average:50,stdev:0|index:1,count:2102953,average:50,stdev:0 GSM2318260_r1 5.04 2.87 0.16 178591879 232197902 165510904 218014528 130.02 131.72 1913722 1630777 220.023 1644.443 92 7413 71.24 77.04 2309995 1363269 2309995 1363269 72.6 73.56 2309995 1389436 2309995 1301638 22468301 12.58 2.01 0 6.85 0 0.10 0 0.09 0 0.00 0 8.81 0 1913722 0 100 0 98.52 0 1.34 0 0.01 0 1.18 0 0.01 0 270.38 0 0.25 0 42360 0 2102953 0 144141 0 2033 0 1857 0 0 0 185341 0 145 0 0 0 1433 0 185455 0 1402 0 188435 0 84.15 0 1769581 0 33195 192805 5.808254255159 2102953.0 1913722.0 42360.0 144141.0 2033.0 1857.0 0.0 185341.0 1769581.0 91.0 2.0 6.9 0.1 0.1 0.0 8.8 84.1 50 50 50.00 38 105147650 27.1 21.9 22.0 29.0 0.0 36.9 23.0 smartseq 1443921 SRR4251042 SRP090061 SRS1698718 SRX2171043 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317261: 26Dp4_C08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317261 GSM2317261 26Dp4_C08_smart-seq 199484000 1994840 2016-09-30 15:56:31 138492312 199484000 1994840 2 1994840 index:0,count:1994840,average:50,stdev:0|index:1,count:1994840,average:50,stdev:0 GSM2317261_r1 2.43 2.8 0.14 172201970 212195990 164664067 204492187 123.23 124.19 1826454 1578948 248.398 2193.066 100 6101 62.84 65.81 2055424 1147718 2055424 1147718 62.57 63.08 2055424 1142787 2055424 1100107 35551934 20.65 1.75 0 4.14 0 0.09 0 0.15 0 0.00 0 8.21 0 1826454 0 100 0 98.71 0 1.40 0 0.01 0 1.24 0 0.01 0 312.24 0 0.26 0 34996 0 1994840 0 82535 0 1714 0 2895 0 0 0 163777 0 128 0 0 0 1227 0 143569 0 1448 0 146372 0 87.42 0 1743919 0 28387 148317 5.224821220981 1994840.0 1826454.0 34996.0 82535.0 1714.0 2895.0 0.0 163777.0 1743919.0 91.6 1.8 4.1 0.1 0.1 0.0 8.2 87.4 50 50 50.00 38 99742000 27.5 21.6 21.7 29.1 0.0 37.3 23.7 smartseq 1443922 SRR4252042 SRP090061 SRS1699718 SRX2172043 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318261: SK_2_G05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318261 GSM2318261 SK_2_G05_smart-seq 39069700 390697 2016-09-30 15:56:31 27589604 39069700 390697 2 390697 index:0,count:390697,average:50,stdev:0|index:1,count:390697,average:50,stdev:0 GSM2318261_r1 0.53 6.19 0.34 767250 581284 662434 499907 75.76 75.47 10207 8530 128.319 16678.893 51 653 11.72 13.99 13873 1196 13873 1196 14.1 13.53 13873 1439 13873 1156 322896 42.08 0.33 0 0.43 0 0.03 0 0.01 0 0.00 0 97.35 0 10207 0 100 0 89.49 0 2.14 0 0.01 0 1.15 0 0.01 0 61.15 0 0.52 0 1298 0 390697 0 1661 0 107 0 37 0 0 0 380346 0 0 0 0 0 0 0 107 0 17 0 124 0 2.19 0 8546 0 82 82 1.000000000000 390697.0 10207.0 1298.0 1661.0 107.0 37.0 0.0 380346.0 8546.0 2.6 0.3 0.4 0.0 0.0 0.0 97.4 2.2 50 50 50.00 38 19534850 21.7 21.7 21.3 35.3 0.0 34.7 15.1 smartseq 1443953 SRR4251043 SRP090061 SRS1698720 SRX2171044 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317262: 26Dp4_C09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317262 GSM2317262 26Dp4_C09_smart-seq 178199900 1781999 2016-09-30 15:56:31 122580478 178199900 1781999 2 1781999 index:0,count:1781999,average:50,stdev:0|index:1,count:1781999,average:50,stdev:0 GSM2317262_r1 1.56 2.85 0.08 155273823 186824886 148783812 180196837 120.32 121.11 1637721 1389062 252.410 2313.634 110 5323 60.54 63.27 1833130 991537 1833130 991537 60.21 60.61 1833130 986023 1833130 949856 34981158 22.53 1.70 0 3.95 0 0.09 0 0.13 0 0.00 0 7.88 0 1637721 0 100 0 98.79 0 1.40 0 0.01 0 1.21 0 0.01 0 256.61 0 0.25 0 30313 0 1781999 0 70459 0 1525 0 2362 0 0 0 140391 0 90 0 0 0 1137 0 141873 0 1461 0 144561 0 87.95 0 1567262 0 30382 148264 4.879994733724 1781999.0 1637721.0 30313.0 70459.0 1525.0 2362.0 0.0 140391.0 1567262.0 91.9 1.7 4.0 0.1 0.1 0.0 7.9 87.9 50 50 50.00 38 89099950 27.5 21.7 21.8 29.0 0.0 37.4 23.9 smartseq 1443954 SRR4252043 SRP090061 SRS1699722 SRX2172044 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318262: SK_2_G06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318262 GSM2318262 SK_2_G06_smart-seq 290332800 2903328 2016-09-30 15:56:31 202563265 290332800 2903328 2 2903328 index:0,count:2903328,average:50,stdev:0|index:1,count:2903328,average:50,stdev:0 GSM2318262_r1 6.6 2.5 0.08 249184531 323539669 229085414 303528871 129.84 132.5 2673035 2258459 220.875 1963.118 81 10374 69.35 75.64 3415256 1853860 3415256 1853860 70.78 72.23 3415256 1892076 3415256 1770306 33216872 13.33 2.03 0 7.65 0 0.10 0 0.12 0 0.00 0 7.71 0 2673035 0 100 0 98.51 0 1.31 0 0.01 0 1.19 0 0.01 0 298.63 0 0.24 0 59065 0 2903328 0 222002 0 2980 0 3416 0 0 0 223897 0 198 0 0 0 2105 0 278575 0 2127 0 283005 0 84.42 0 2451033 0 40388 285588 7.071110230762 2903328.0 2673035.0 59065.0 222002.0 2980.0 3416.0 0.0 223897.0 2451033.0 92.1 2.0 7.6 0.1 0.1 0.0 7.7 84.4 50 50 50.00 38 145166400 26.7 22.5 22.6 28.2 0.0 37.3 24.3 smartseq 1443985 SRR4251044 SRP090061 SRS1698721 SRX2171045 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317263: 26Dp4_C10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317263 GSM2317263 26Dp4_C10_smart-seq 180186300 1801863 2016-09-30 15:56:31 124823212 180186300 1801863 2 1801863 index:0,count:1801863,average:50,stdev:0|index:1,count:1801863,average:50,stdev:0 GSM2317263_r1 0.45 2.97 0.08 150213320 176906458 145323598 172052001 117.77 118.39 1590890 1405466 248.339 1869.624 110 5542 54.14 56.01 1731087 861317 1731087 861317 53.69 54.04 1731087 854210 1731087 831006 41191993 27.42 1.70 0 2.95 0 0.09 0 0.16 0 0.00 0 11.46 0 1590890 0 100 0 98.78 0 1.41 0 0.01 0 1.21 0 0.01 0 231.67 0 0.25 0 30624 0 1801863 0 53197 0 1647 0 2900 0 0 0 206426 0 58 0 0 0 1089 0 98466 0 1642 0 101255 0 85.34 0 1537693 0 20395 102937 5.047168423633 1801863.0 1590890.0 30624.0 53197.0 1647.0 2900.0 0.0 206426.0 1537693.0 88.3 1.7 3.0 0.1 0.2 0.0 11.5 85.3 50 50 50.00 38 90093150 28.0 20.9 21.0 30.1 0.0 37.3 23.2 smartseq 1443986 SRR4252044 SRP090061 SRS1699720 SRX2172045 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318263: SK_2_G07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318263 GSM2318263 SK_2_G07_smart-seq 229492100 2294921 2016-09-30 15:56:31 160384131 229492100 2294921 2 2294921 index:0,count:2294921,average:50,stdev:0|index:1,count:2294921,average:50,stdev:0 GSM2318263_r1 3.29 3.03 0.12 197647373 249997820 185647099 237960344 126.49 128.18 2108809 1795657 231.949 2031.341 110 7517 66.74 71.19 2517360 1407363 2517360 1407363 66.99 67.93 2517360 1412596 2517360 1342862 32709804 16.55 1.87 0 5.75 0 0.10 0 0.11 0 0.00 0 7.90 0 2108809 0 100 0 98.65 0 1.38 0 0.01 0 1.22 0 0.01 0 275.39 0 0.24 0 42948 0 2294921 0 131981 0 2305 0 2554 0 0 0 181253 0 171 0 0 0 1727 0 192500 0 1754 0 196152 0 86.14 0 1976828 0 42022 200258 4.765551377850 2294921.0 2108809.0 42948.0 131981.0 2305.0 2554.0 0.0 181253.0 1976828.0 91.9 1.9 5.8 0.1 0.1 0.0 7.9 86.1 50 50 50.00 38 114746050 27.5 21.6 21.8 29.1 0.0 37.2 24.0 smartseq 1444018 SRR4251045 SRP090061 SRS1698724 SRX2171046 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317264: 26Dp4_C11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317264 GSM2317264 26Dp4_C11_smart-seq 229987700 2299877 2016-09-30 15:56:31 159790191 229987700 2299877 2 2299877 index:0,count:2299877,average:50,stdev:0|index:1,count:2299877,average:50,stdev:0 GSM2317264_r1 1.14 2.96 0.09 201126068 247687996 191494036 237978954 123.15 124.27 2107362 1754079 261.554 2525.547 110 6519 65.75 69.13 2411477 1385489 2411477 1385489 65.65 66.36 2411477 1383522 2411477 1330068 37116299 18.45 1.70 0 4.48 0 0.11 0 0.08 0 0.00 0 8.18 0 2107362 0 100 0 98.77 0 1.41 0 0.01 0 1.21 0 0.01 0 285.50 0 0.27 0 39095 0 2299877 0 103122 0 2538 0 1859 0 0 0 188118 0 138 0 0 0 1594 0 195529 0 1820 0 199081 0 87.15 0 2004240 0 30420 204661 6.727843523997 2299877.0 2107362.0 39095.0 103122.0 2538.0 1859.0 0.0 188118.0 2004240.0 91.6 1.7 4.5 0.1 0.1 0.0 8.2 87.1 50 50 50.00 38 114993850 27.3 21.8 21.9 28.9 0.0 37.2 23.5 smartseq 1444019 SRR4252045 SRP090061 SRS1699727 SRX2172046 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318264: SK_2_G08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318264 GSM2318264 SK_2_G08_smart-seq 243035400 2430354 2016-09-30 15:56:31 169277075 243035400 2430354 2 2430354 index:0,count:2430354,average:50,stdev:0|index:1,count:2430354,average:50,stdev:0 GSM2318264_r1 3.88 3.35 0.1 208549965 265124633 194889063 251107494 127.13 128.85 2233253 1865991 220.620 1985.873 88 8587 71.57 76.75 2668993 1598409 2668993 1598409 72.61 73.69 2668993 1621645 2668993 1534810 26217667 12.57 1.91 0 6.19 0 0.12 0 0.11 0 0.00 0 7.88 0 2233253 0 100 0 98.55 0 1.34 0 0.01 0 1.19 0 0.01 0 336.51 0 0.23 0 46389 0 2430354 0 150534 0 2797 0 2794 0 0 0 191510 0 183 0 0 0 2061 0 242186 0 1778 0 246208 0 85.70 0 2082719 0 40456 249340 6.163239074550 2430354.0 2233253.0 46389.0 150534.0 2797.0 2794.0 0.0 191510.0 2082719.0 91.9 1.9 6.2 0.1 0.1 0.0 7.9 85.7 50 50 50.00 38 121517700 27.1 22.1 22.2 28.7 0.0 37.3 24.0 smartseq 1444048 SRR4251046 SRP090061 SRS1698723 SRX2171047 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317265: 26Dp4_D01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317265 GSM2317265 26Dp4_D01_smart-seq 189725400 1897254 2016-09-30 15:56:31 133708181 189725400 1897254 2 1897254 index:0,count:1897254,average:50,stdev:0|index:1,count:1897254,average:50,stdev:0 GSM2317265_r1 2.32 3.08 0.11 164793633 203739962 156498564 195038105 123.63 124.63 1737871 1449005 253.409 2230.700 100 5615 66.42 70.05 1986357 1154301 1986357 1154301 66.73 67.26 1986357 1159764 1986357 1108448 29900463 18.14 1.84 0 4.74 0 0.07 0 0.12 0 0.00 0 8.21 0 1737871 0 100 0 98.71 0 1.38 0 0.01 0 1.21 0 0.01 0 227.67 0 0.28 0 34928 0 1897254 0 89962 0 1264 0 2274 0 0 0 155845 0 121 0 0 0 1600 0 166907 0 1434 0 170062 0 86.86 0 1647909 0 29638 173999 5.870807746812 1897254.0 1737871.0 34928.0 89962.0 1264.0 2274.0 0.0 155845.0 1647909.0 91.6 1.8 4.7 0.1 0.1 0.0 8.2 86.9 50 50 50.00 38 94862700 27.4 21.7 21.9 28.9 0.0 37.1 23.6 smartseq 1444049 SRR4252046 SRP090061 SRS1699721 SRX2172047 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318265: SK_2_G09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318265 GSM2318265 SK_2_G09_smart-seq 218168400 2181684 2016-09-30 15:56:31 150907193 218168400 2181684 2 2181684 index:0,count:2181684,average:50,stdev:0|index:1,count:2181684,average:50,stdev:0 GSM2318265_r1 4.27 2.84 0.09 185500337 241078702 174082003 228566529 129.96 131.3 1996045 1690346 217.673 1914.509 80 7902 74.26 79.31 2345913 1482200 2345913 1482200 74.4 75.42 2345913 1485118 2345913 1409637 21399660 11.54 1.91 0 5.83 0 0.08 0 0.09 0 0.00 0 8.34 0 1996045 0 100 0 98.52 0 1.36 0 0.01 0 1.20 0 0.01 0 253.36 0 0.22 0 41738 0 2181684 0 127092 0 1656 0 1950 0 0 0 182033 0 163 0 0 0 1941 0 201870 0 1564 0 205538 0 85.67 0 1868953 0 34166 208526 6.103319089153 2181684.0 1996045.0 41738.0 127092.0 1656.0 1950.0 0.0 182033.0 1868953.0 91.5 1.9 5.8 0.1 0.1 0.0 8.3 85.7 50 50 50.00 38 109084200 27.0 22.1 22.2 28.7 0.0 37.3 23.8 smartseq 1444081 SRR4251047 SRP090061 SRS1698722 SRX2171048 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317266: 26Dp4_D02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317266 GSM2317266 26Dp4_D02_smart-seq 182685000 1826850 2016-09-30 15:56:31 129296543 182685000 1826850 2 1826850 index:0,count:1826850,average:50,stdev:0|index:1,count:1826850,average:50,stdev:0 GSM2317266_r1 2.41 3.14 0.15 161204068 205758321 152312633 196327978 127.64 128.9 1682551 1379924 268.176 2589.839 137 4902 69.87 74.04 1947192 1175654 1947192 1175654 69.74 70.63 1947192 1173482 1947192 1121461 25106548 15.57 1.79 0 5.18 0 0.09 0 0.10 0 0.00 0 7.71 0 1682551 0 100 0 98.73 0 1.38 0 0.01 0 1.21 0 0.01 0 346.14 0 0.29 0 32664 0 1826850 0 94700 0 1594 0 1908 0 0 0 140797 0 106 0 0 0 1393 0 165243 0 1377 0 168119 0 86.92 0 1587851 0 33725 172451 5.113446997776 1826850.0 1682551.0 32664.0 94700.0 1594.0 1908.0 0.0 140797.0 1587851.0 92.1 1.8 5.2 0.1 0.1 0.0 7.7 86.9 50 50 50.00 38 91342500 27.2 22.0 22.3 28.5 0.0 37.1 23.5 smartseq 1444082 SRR4252047 SRP090061 SRS1699723 SRX2172048 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318266: SK_2_G10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318266 GSM2318266 SK_2_G10_smart-seq 233230300 2332303 2016-09-30 15:56:31 161356693 233230300 2332303 2 2332303 index:0,count:2332303,average:50,stdev:0|index:1,count:2332303,average:50,stdev:0 GSM2318266_r1 7.21 2.96 0.14 193846793 257248858 176643730 238208623 132.71 134.85 2123089 1835482 186.643 1373.956 83 10450 74.29 81.78 2647721 1577152 2647721 1577152 76.81 78.03 2647721 1630811 2647721 1504750 18950564 9.78 2.41 0 8.35 0 0.09 0 0.08 0 0.00 0 8.80 0 2123089 0 100 0 98.27 0 1.30 0 0.01 0 1.19 0 0.01 0 246.95 0 0.23 0 56216 0 2332303 0 194661 0 2069 0 1809 0 0 0 205336 0 198 0 0 0 1726 0 239554 0 1586 0 243064 0 82.68 0 1928428 0 41201 246162 5.974660809204 2332303.0 2123089.0 56216.0 194661.0 2069.0 1809.0 0.0 205336.0 1928428.0 91.0 2.4 8.3 0.1 0.1 0.0 8.8 82.7 50 50 50.00 38 116615150 26.9 22.2 22.3 28.7 0.0 37.3 23.9 smartseq 1444113 SRR4251048 SRP090061 SRS1698725 SRX2171049 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317267: 26Dp4_D03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317267 GSM2317267 26Dp4_D03_smart-seq 149062400 1490624 2016-09-30 15:56:31 106019858 149062400 1490624 2 1490624 index:0,count:1490624,average:50,stdev:0|index:1,count:1490624,average:50,stdev:0 GSM2317267_r1 5.88 3.48 0.14 130673512 168711350 124236444 161794685 129.11 130.23 1368030 1109709 269.562 2920.826 115 3944 73.29 77.2 1553172 1002623 1553172 1002623 73.3 74.11 1553172 1002704 1553172 962474 17637695 13.50 1.82 0 4.65 0 0.09 0 0.09 0 0.00 0 8.05 0 1368030 0 100 0 98.68 0 1.36 0 0.01 0 1.22 0 0.01 0 243.92 0 0.31 0 27093 0 1490624 0 69310 0 1294 0 1307 0 0 0 119993 0 163 0 0 0 1107 0 141150 0 1056 0 143476 0 87.13 0 1298720 0 36813 147074 3.995164751582 1490624.0 1368030.0 27093.0 69310.0 1294.0 1307.0 0.0 119993.0 1298720.0 91.8 1.8 4.6 0.1 0.1 0.0 8.0 87.1 50 50 50.00 38 74531200 27.2 22.0 22.3 28.5 0.0 37.1 23.5 smartseq 1444114 SRR4252048 SRP090061 SRS1699726 SRX2172049 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318267: SK_2_G11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318267 GSM2318267 SK_2_G11_smart-seq 253043100 2530431 2016-09-30 15:56:31 175655091 253043100 2530431 2 2530431 index:0,count:2530431,average:50,stdev:0|index:1,count:2530431,average:50,stdev:0 GSM2318267_r1 1.99 3.19 0.08 211586032 266459553 197494214 252209482 125.93 127.7 2278275 1935327 212.075 1782.374 100 9400 71.79 77.06 2755229 1635678 2755229 1635678 72.44 73.68 2755229 1650363 2755229 1563881 27756145 13.12 1.93 0 6.16 0 0.09 0 0.09 0 0.00 0 9.79 0 2278275 0 100 0 98.51 0 1.38 0 0.01 0 1.19 0 0.01 0 325.34 0 0.22 0 48794 0 2530431 0 155762 0 2164 0 2222 0 0 0 247770 0 197 0 0 0 1713 0 236268 0 1583 0 239761 0 83.88 0 2122513 0 32679 244722 7.488662443771 2530431.0 2278275.0 48794.0 155762.0 2164.0 2222.0 0.0 247770.0 2122513.0 90.0 1.9 6.2 0.1 0.1 0.0 9.8 83.9 50 50 50.00 38 126521550 27.1 21.8 21.9 29.2 0.0 37.2 23.3 smartseq 1444144 SRR4251049 SRP090061 SRS1698728 SRX2171050 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317268: 26Dp4_D04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317268 GSM2317268 26Dp4_D04_smart-seq 183594100 1835941 2016-09-30 15:56:31 128988274 183594100 1835941 2 1835941 index:0,count:1835941,average:50,stdev:0|index:1,count:1835941,average:50,stdev:0 GSM2317268_r1 2.57 2.76 0.16 160281543 199686418 152887105 191884154 124.58 125.51 1684994 1427099 249.023 2271.164 110 5578 66.42 69.72 1902717 1119170 1902717 1119170 66.53 67.1 1902717 1121052 1902717 1077217 28777332 17.95 1.73 0 4.34 0 0.10 0 0.12 0 0.00 0 8.00 0 1684994 0 100 0 98.74 0 1.36 0 0.01 0 1.22 0 0.01 0 236.05 0 0.28 0 31723 0 1835941 0 79697 0 1828 0 2292 0 0 0 146827 0 105 0 0 0 1277 0 146950 0 1304 0 149636 0 87.44 0 1605297 0 28924 153682 5.313303830729 1835941.0 1684994.0 31723.0 79697.0 1828.0 2292.0 0.0 146827.0 1605297.0 91.8 1.7 4.3 0.1 0.1 0.0 8.0 87.4 50 50 50.00 38 91797050 27.4 21.7 21.8 29.0 0.0 37.1 23.5 smartseq 1444145 SRR4252049 SRP090061 SRS1699724 SRX2172050 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318268: SK_2_G12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318268 GSM2318268 SK_2_G12_smart-seq 210892200 2108922 2016-09-30 15:56:31 146931937 210892200 2108922 2 2108922 index:0,count:2108922,average:50,stdev:0|index:1,count:2108922,average:50,stdev:0 GSM2318268_r1 4.17 2.86 0.14 178021552 229556810 164864799 215131513 128.95 130.49 1920445 1601951 218.750 1817.435 78 7561 72.94 78.96 2310858 1400718 2310858 1400718 74.42 75.28 2310858 1429152 2310858 1335531 20233587 11.37 2.20 0 6.94 0 0.08 0 0.08 0 0.00 0 8.78 0 1920445 0 100 0 98.42 0 1.33 0 0.01 0 1.21 0 0.01 0 330.09 0 0.23 0 46300 0 2108922 0 146456 0 1649 0 1608 0 0 0 185220 0 191 0 0 0 1555 0 213986 0 1318 0 217050 0 84.12 0 1773989 0 36198 218988 6.049726504227 2108922.0 1920445.0 46300.0 146456.0 1649.0 1608.0 0.0 185220.0 1773989.0 91.1 2.2 6.9 0.1 0.1 0.0 8.8 84.1 50 50 50.00 38 105446100 26.9 22.1 22.2 28.7 0.0 37.3 23.9 smartseq 1444369 SRR4251050 SRP090061 SRS1698727 SRX2171051 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317269: 26Dp4_D05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317269 GSM2317269 26Dp4_D05_smart-seq 209416700 2094167 2016-09-30 15:56:31 147092690 209416700 2094167 2 2094167 index:0,count:2094167,average:50,stdev:0|index:1,count:2094167,average:50,stdev:0 GSM2317269_r1 3.22 2.7 0.11 169515492 209567927 160512576 200574132 123.63 124.96 1788174 1514235 253.607 2344.568 103 5703 64.2 67.94 2079847 1148034 2079847 1148034 64.28 65.06 2079847 1149391 2079847 1099238 33283974 19.63 1.69 0 4.70 0 0.07 0 0.11 0 0.00 0 14.43 0 1788174 0 100 0 98.75 0 1.37 0 0.01 0 1.21 0 0.01 0 221.74 0 0.28 0 35363 0 2094167 0 98512 0 1510 0 2329 0 0 0 302154 0 122 0 0 0 1203 0 154989 0 1343 0 157657 0 80.68 0 1689662 0 31772 160799 5.061028578623 2094167.0 1788174.0 35363.0 98512.0 1510.0 2329.0 0.0 302154.0 1689662.0 85.4 1.7 4.7 0.1 0.1 0.0 14.4 80.7 50 50 50.00 38 104708350 27.3 21.8 22.0 28.8 0.0 37.1 23.3 smartseq 1444370 SRR4252050 SRP090061 SRS1699725 SRX2172051 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318269: SK_2_H01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318269 GSM2318269 SK_2_H01_smart-seq 150177600 1501776 2016-09-30 15:56:31 106030429 150177600 1501776 2 1501776 index:0,count:1501776,average:50,stdev:0|index:1,count:1501776,average:50,stdev:0 GSM2318269_r1 15.44 2.1 0.08 127200918 176139548 116731520 166495416 138.47 142.63 1372411 1201471 224.743 1799.201 69 5225 72.69 79.55 1841134 997595 1841134 997595 73.6 76.4 1841134 1010040 1841134 958079 14220019 11.18 2.07 0 7.89 0 0.11 0 0.07 0 0.00 0 8.43 0 1372411 0 100 0 98.29 0 1.29 0 0.01 0 1.20 0 0.01 0 337.90 0 0.27 0 31095 0 1501776 0 118432 0 1711 0 990 0 0 0 126664 0 107 0 0 0 802 0 103836 0 965 0 105710 0 83.50 0 1253979 0 36135 104585 2.894285318943 1501776.0 1372411.0 31095.0 118432.0 1711.0 990.0 0.0 126664.0 1253979.0 91.4 2.1 7.9 0.1 0.1 0.0 8.4 83.5 50 50 50.00 38 75088800 27.1 21.9 22.1 29.0 0.0 37.0 23.2 smartseq 1444400 SRR4251051 SRP090061 SRS1698726 SRX2171052 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317270: 26Dp4_D06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317270 GSM2317270 26Dp4_D06_smart-seq 196159500 1961595 2016-09-30 15:56:31 138138816 196159500 1961595 2 1961595 index:0,count:1961595,average:50,stdev:0|index:1,count:1961595,average:50,stdev:0 GSM2317270_r1 2.12 3.16 0.17 170107306 212174065 160647803 202312223 124.73 125.94 1804844 1519436 246.713 2415.070 100 6009 66.64 70.69 2102747 1202836 2102747 1202836 66.49 67.25 2102747 1199980 2102747 1144304 29437631 17.31 1.84 0 5.27 0 0.09 0 0.13 0 0.00 0 7.77 0 1804844 0 100 0 98.67 0 1.36 0 0.01 0 1.24 0 0.01 0 243.51 0 0.28 0 36044 0 1961595 0 103344 0 1722 0 2599 0 0 0 152430 0 123 0 0 0 1236 0 168473 0 1467 0 171299 0 86.74 0 1701500 0 33430 175178 5.240143583608 1961595.0 1804844.0 36044.0 103344.0 1722.0 2599.0 0.0 152430.0 1701500.0 92.0 1.8 5.3 0.1 0.1 0.0 7.8 86.7 50 50 50.00 38 98079750 27.2 22.0 22.2 28.6 0.0 37.1 23.7 smartseq 1444401 SRR4252051 SRP090061 SRS1699728 SRX2172052 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318270: SK_2_H02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318270 GSM2318270 SK_2_H02_smart-seq 217207600 2172076 2016-09-30 15:56:31 152249957 217207600 2172076 2 2172076 index:0,count:2172076,average:50,stdev:0|index:1,count:2172076,average:50,stdev:0 GSM2318270_r1 7.19 2.94 0.11 184939762 247865000 169463070 230721038 134.02 136.15 1989814 1637704 229.469 2023.336 82 7228 75.04 82.15 2459732 1493172 2459732 1493172 77.41 78.61 2459732 1540275 2459732 1428898 17581683 9.51 2.29 0 7.92 0 0.08 0 0.09 0 0.00 0 8.22 0 1989814 0 100 0 98.35 0 1.30 0 0.01 0 1.21 0 0.01 0 260.65 0 0.25 0 49760 0 2172076 0 172126 0 1638 0 1998 0 0 0 178626 0 130 0 0 0 1497 0 229424 0 1555 0 232606 0 83.68 0 1817688 0 42057 235647 5.603038733148 2172076.0 1989814.0 49760.0 172126.0 1638.0 1998.0 0.0 178626.0 1817688.0 91.6 2.3 7.9 0.1 0.1 0.0 8.2 83.7 50 50 50.00 38 108603800 26.8 22.4 22.5 28.3 0.0 37.2 24.1 smartseq 1444433 SRR4251052 SRP090061 SRS1698729 SRX2171053 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317271: 26Dp4_D07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317271 GSM2317271 26Dp4_D07_smart-seq 201453700 2014537 2016-09-30 15:56:31 141925998 201453700 2014537 2 2014537 index:0,count:2014537,average:50,stdev:0|index:1,count:2014537,average:50,stdev:0 GSM2317271_r1 3.86 3.16 0.18 175144486 221238213 164932882 210631305 126.32 127.71 1846425 1542400 252.831 2336.857 113 5850 67.4 71.7 2156009 1244416 2156009 1244416 67.98 68.59 2156009 1255148 2156009 1190519 28629872 16.35 1.92 0 5.50 0 0.06 0 0.09 0 0.00 0 8.19 0 1846425 0 100 0 98.67 0 1.37 0 0.01 0 1.22 0 0.01 0 259.01 0 0.28 0 38587 0 2014537 0 110769 0 1278 0 1881 0 0 0 164953 0 88 0 0 0 1450 0 177081 0 1474 0 180093 0 86.16 0 1735656 0 32195 183490 5.699332194440 2014537.0 1846425.0 38587.0 110769.0 1278.0 1881.0 0.0 164953.0 1735656.0 91.7 1.9 5.5 0.1 0.1 0.0 8.2 86.2 50 50 50.00 38 100726850 27.3 21.9 22.1 28.7 0.0 37.1 23.6 smartseq 1444434 SRR4252052 SRP090061 SRS1699731 SRX2172053 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318271: SK_2_H03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318271 GSM2318271 SK_2_H03_smart-seq 223039700 2230397 2016-09-30 15:56:31 156065521 223039700 2230397 2 2230397 index:0,count:2230397,average:50,stdev:0|index:1,count:2230397,average:50,stdev:0 GSM2318271_r1 4.93 2.42 0.1 166517619 218665526 152660935 203141916 131.32 133.07 1843990 1551744 189.559 1563.296 78 9283 89.63 98.08 2300043 1652746 2300043 1652746 91.65 94.25 2300043 1690098 2300043 1588287 1151410 0.69 2.39 0 7.12 0 0.09 0 0.01 0 0.00 0 17.23 0 1843990 0 100 0 97.87 0 1.39 0 0.01 0 1.14 0 0.01 0 308.82 0 0.23 0 53285 0 2230397 0 158832 0 1903 0 219 0 0 0 384285 0 168 0 0 0 2446 0 256953 0 1556 0 261123 0 75.55 0 1685158 0 20257 263579 13.011749025028 2230397.0 1843990.0 53285.0 158832.0 1903.0 219.0 0.0 384285.0 1685158.0 82.7 2.4 7.1 0.1 0.0 0.0 17.2 75.6 50 50 50.00 38 111519850 25.7 22.7 22.7 28.9 0.0 37.0 21.5 smartseq 1444465 SRR4251053 SRP090061 SRS1698730 SRX2171054 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317272: 26Dp4_D08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317272 GSM2317272 26Dp4_D08_smart-seq 143774500 1437745 2016-09-30 15:56:31 101972167 143774500 1437745 2 1437745 index:0,count:1437745,average:50,stdev:0|index:1,count:1437745,average:50,stdev:0 GSM2317272_r1 8.47 2.66 0.15 120766688 162855018 110268650 152119474 134.85 137.95 1278372 1012343 252.619 2395.578 105 3877 85.46 93.88 1636770 1092541 1636770 1092541 87.17 89.85 1636770 1114346 1636770 1045668 3197550 2.65 2.19 0 7.97 0 0.05 0 0.02 0 0.00 0 11.02 0 1278372 0 100 0 98.19 0 1.29 0 0.01 0 1.19 0 0.01 0 215.66 0 0.30 0 31461 0 1437745 0 114579 0 694 0 294 0 0 0 158385 0 133 0 0 0 1227 0 153520 0 790 0 155670 0 80.95 0 1163793 0 27225 158172 5.809807162534 1437745.0 1278372.0 31461.0 114579.0 694.0 294.0 0.0 158385.0 1163793.0 88.9 2.2 8.0 0.0 0.0 0.0 11.0 80.9 50 50 50.00 38 71887250 26.5 22.8 23.2 27.6 0.0 37.1 23.5 smartseq 1444466 SRR4252053 SRP090061 SRS1699732 SRX2172054 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318272: SK_2_H04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318272 GSM2318272 SK_2_H04_smart-seq 243204000 2432040 2016-09-30 15:56:31 169057104 243204000 2432040 2 2432040 index:0,count:2432040,average:50,stdev:0|index:1,count:2432040,average:50,stdev:0 GSM2318272_r1 3.28 2.76 0.1 206201234 263017009 193900514 249629190 127.55 128.74 2224430 1892217 213.290 1918.014 83 8877 70.87 75.51 2591350 1576344 2591350 1576344 71.54 72.22 2591350 1591425 2591350 1507755 29245244 14.18 1.93 0 5.63 0 0.08 0 0.08 0 0.00 0 8.38 0 2224430 0 100 0 98.51 0 1.34 0 0.01 0 1.21 0 0.01 0 291.84 0 0.22 0 46895 0 2432040 0 136837 0 1856 0 2011 0 0 0 203743 0 164 0 0 0 1695 0 220743 0 1712 0 224314 0 85.84 0 2087593 0 35206 228938 6.502812020678 2432040.0 2224430.0 46895.0 136837.0 1856.0 2011.0 0.0 203743.0 2087593.0 91.5 1.9 5.6 0.1 0.1 0.0 8.4 85.8 50 50 50.00 38 121602000 27.3 21.7 21.8 29.2 0.0 37.2 23.6 smartseq 1444498 SRR4251054 SRP090061 SRS1698731 SRX2171055 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317273: 26Dp4_D09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317273 GSM2317273 26Dp4_D09_smart-seq 103288100 1032881 2016-09-30 15:56:31 72407638 103288100 1032881 2 1032881 index:0,count:1032881,average:50,stdev:0|index:1,count:1032881,average:50,stdev:0 GSM2317273_r1 1.45 3.1 0.16 89978717 108937695 86141846 105206020 121.07 122.13 943850 800663 265.261 2447.941 110 2829 61.45 64.27 1070940 580042 1070940 580042 60.97 61.57 1070940 575420 1070940 555607 19031742 21.15 1.73 0 4.01 0 0.09 0 0.13 0 0.00 0 8.40 0 943850 0 100 0 98.68 0 1.43 0 0.01 0 1.22 0 0.01 0 218.73 0 0.29 0 17849 0 1032881 0 41412 0 895 0 1337 0 0 0 86799 0 41 0 0 0 673 0 76619 0 744 0 78077 0 87.37 0 902438 0 24118 79142 3.281449539763 1032881.0 943850.0 17849.0 41412.0 895.0 1337.0 0.0 86799.0 902438.0 91.4 1.7 4.0 0.1 0.1 0.0 8.4 87.4 50 50 50.00 38 51644050 27.6 21.5 21.8 29.0 0.0 37.2 23.9 smartseq 1444499 SRR4252054 SRP090061 SRS1699730 SRX2172055 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318273: SK_2_H05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318273 GSM2318273 SK_2_H05_smart-seq 240942200 2409422 2016-09-30 15:56:31 167715542 240942200 2409422 2 2409422 index:0,count:2409422,average:50,stdev:0|index:1,count:2409422,average:50,stdev:0 GSM2318273_r1 4.26 2.81 0.1 203983763 264585618 191838125 251269327 129.71 130.98 2196306 1864854 220.748 1898.986 81 8438 73.78 78.64 2567306 1620490 2567306 1620490 73.88 74.84 2567306 1622661 2567306 1542243 24447333 11.98 1.95 0 5.63 0 0.08 0 0.08 0 0.00 0 8.68 0 2196306 0 100 0 98.50 0 1.36 0 0.01 0 1.21 0 0.01 0 279.80 0 0.22 0 47021 0 2409422 0 135570 0 1905 0 2039 0 0 0 209172 0 150 0 0 0 2018 0 216169 0 1651 0 219988 0 85.53 0 2060736 0 34631 223144 6.443475498831 2409422.0 2196306.0 47021.0 135570.0 1905.0 2039.0 0.0 209172.0 2060736.0 91.2 2.0 5.6 0.1 0.1 0.0 8.7 85.5 50 50 50.00 38 120471100 27.2 21.8 21.9 29.1 0.0 37.2 23.6 smartseq 1444531 SRR4251055 SRP090061 SRS1698734 SRX2171056 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317274: 26Dp4_D10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317274 GSM2317274 26Dp4_D10_smart-seq 202558100 2025581 2016-09-30 15:56:31 142792087 202558100 2025581 2 2025581 index:0,count:2025581,average:50,stdev:0|index:1,count:2025581,average:50,stdev:0 GSM2317274_r1 2.42 3.43 0.26 171644398 213838621 163011293 204875466 124.58 125.68 1828356 1541107 242.269 2327.902 83 6206 67.2 70.88 2091457 1228716 2091457 1228716 66.98 67.71 2091457 1224578 2091457 1173742 30464178 17.75 1.77 0 4.68 0 0.08 0 0.09 0 0.00 0 9.57 0 1828356 0 100 0 98.64 0 1.36 0 0.01 0 1.22 0 0.01 0 291.68 0 0.28 0 35897 0 2025581 0 94893 0 1528 0 1859 0 0 0 193838 0 160 0 0 0 1370 0 168788 0 1525 0 171843 0 85.58 0 1733463 0 30232 175170 5.794191585075 2025581.0 1828356.0 35897.0 94893.0 1528.0 1859.0 0.0 193838.0 1733463.0 90.3 1.8 4.7 0.1 0.1 0.0 9.6 85.6 50 50 50.00 38 101279050 27.3 21.8 21.9 29.0 0.0 37.1 23.4 smartseq 1444532 SRR4252055 SRP090061 SRS1699729 SRX2172056 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318274: SK_2_H06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318274 GSM2318274 SK_2_H06_smart-seq 256880100 2568801 2016-09-30 15:56:31 178541764 256880100 2568801 2 2568801 index:0,count:2568801,average:50,stdev:0|index:1,count:2568801,average:50,stdev:0 GSM2318274_r1 3.42 3.07 0.2 214592820 274651070 202083770 260938840 127.99 129.12 2335171 2019958 201.316 1761.787 83 10282 72.29 76.93 2719714 1688064 2719714 1688064 72.69 73.61 2719714 1697550 2719714 1615091 28850754 13.44 2.00 0 5.49 0 0.08 0 0.08 0 0.00 0 8.94 0 2335171 0 100 0 98.45 0 1.33 0 0.01 0 1.20 0 0.01 0 420.35 0 0.23 0 51307 0 2568801 0 140970 0 1964 0 2042 0 0 0 229624 0 161 0 0 0 1594 0 226712 0 1931 0 230398 0 85.42 0 2194201 0 33125 234344 7.074535849057 2568801.0 2335171.0 51307.0 140970.0 1964.0 2042.0 0.0 229624.0 2194201.0 90.9 2.0 5.5 0.1 0.1 0.0 8.9 85.4 50 50 50.00 38 128440050 27.3 21.6 21.7 29.3 0.0 37.2 23.3 smartseq 1444563 SRR4251056 SRP090061 SRS1698732 SRX2171057 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317275: 26Dp4_D11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317275 GSM2317275 26Dp4_D11_smart-seq 198924800 1989248 2016-09-30 15:56:31 139867480 198924800 1989248 2 1989248 index:0,count:1989248,average:50,stdev:0|index:1,count:1989248,average:50,stdev:0 GSM2317275_r1 2.96 3.02 0.08 164608686 207395067 155635084 197655915 125.99 127.0 1749973 1461812 241.020 2225.756 81 5985 68.21 72.27 2014148 1193643 2014148 1193643 68.52 69.04 2014148 1199063 2014148 1140282 28065597 17.05 1.83 0 4.95 0 0.07 0 0.12 0 0.00 0 11.84 0 1749973 0 100 0 98.63 0 1.36 0 0.01 0 1.21 0 0.01 0 255.76 0 0.29 0 36471 0 1989248 0 98433 0 1368 0 2328 0 0 0 235579 0 134 0 0 0 1454 0 175797 0 1319 0 178704 0 83.02 0 1651540 0 34970 181647 5.194366599943 1989248.0 1749973.0 36471.0 98433.0 1368.0 2328.0 0.0 235579.0 1651540.0 88.0 1.8 4.9 0.1 0.1 0.0 11.8 83.0 50 50 50.00 38 99462400 27.0 22.2 22.4 28.4 0.0 37.1 23.5 smartseq 1444564 SRR4252056 SRP090061 SRS1699733 SRX2172057 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318275: SK_2_H07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318275 GSM2318275 SK_2_H07_smart-seq 130489100 1304891 2016-09-30 15:56:31 92027971 130489100 1304891 2 1304891 index:0,count:1304891,average:50,stdev:0|index:1,count:1304891,average:50,stdev:0 GSM2318275_r1 15.99 2.17 0.07 111016056 154294195 102423263 146159116 138.98 142.7 1192455 1035877 238.715 1948.318 81 4184 73.23 79.71 1563707 873176 1563707 873176 74.02 76.57 1563707 882637 1563707 838743 12465722 11.23 2.10 0 7.44 0 0.10 0 0.06 0 0.00 0 8.45 0 1192455 0 100 0 98.34 0 1.30 0 0.01 0 1.20 0 0.01 0 223.70 0 0.27 0 27460 0 1304891 0 97021 0 1359 0 833 0 0 0 110244 0 50 0 0 0 785 0 90716 0 812 0 92363 0 83.95 0 1095434 0 33253 91365 2.747571647671 1304891.0 1192455.0 27460.0 97021.0 1359.0 833.0 0.0 110244.0 1095434.0 91.4 2.1 7.4 0.1 0.1 0.0 8.4 83.9 50 50 50.00 38 65244550 27.0 22.0 22.2 28.8 0.0 37.0 23.4 smartseq 1444595 SRR4251057 SRP090061 SRS1698733 SRX2171058 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317276: 26Dp4_E01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317276 GSM2317276 26Dp4_E01_smart-seq 257084600 2570846 2016-09-30 15:56:31 179131105 257084600 2570846 2 2570846 index:0,count:2570846,average:50,stdev:0|index:1,count:2570846,average:50,stdev:0 GSM2317276_r1 0.42 3.02 0.16 226021138 275304960 217162170 266092783 121.8 122.53 2371539 1967958 258.437 2492.189 100 7408 67.4 70.22 2622042 1598532 2622042 1598532 66.87 67.41 2622042 1585798 2622042 1534387 41334232 18.29 1.69 0 3.70 0 0.12 0 0.14 0 0.00 0 7.50 0 2371539 0 100 0 98.77 0 1.42 0 0.01 0 1.22 0 0.01 0 342.78 0 0.26 0 43545 0 2570846 0 95196 0 3102 0 3520 0 0 0 192685 0 157 0 0 0 1803 0 228494 0 2083 0 232537 0 88.54 0 2276343 0 32844 237699 7.237212276215 2570846.0 2371539.0 43545.0 95196.0 3102.0 3520.0 0.0 192685.0 2276343.0 92.2 1.7 3.7 0.1 0.1 0.0 7.5 88.5 50 50 50.00 38 128542300 27.4 21.7 21.9 28.9 0.0 37.2 23.6 smartseq 1444596 SRR4252057 SRP090061 SRS1699737 SRX2172058 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318276: SK_2_H08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318276 GSM2318276 SK_2_H08_smart-seq 229298700 2292987 2016-09-30 15:56:31 160284432 229298700 2292987 2 2292987 index:0,count:2292987,average:50,stdev:0|index:1,count:2292987,average:50,stdev:0 GSM2318276_r1 2.54 2.85 0.1 197640066 259359665 186541176 248081074 131.23 132.99 2120918 1732101 223.474 2008.355 110 8060 76.06 80.75 2502703 1613218 2502703 1613218 76.12 77.38 2502703 1614451 2502703 1545929 21934359 11.10 2.03 0 5.36 0 0.08 0 0.08 0 0.00 0 7.35 0 2120918 0 100 0 98.44 0 1.36 0 0.01 0 1.19 0 0.01 0 242.79 0 0.24 0 46524 0 2292987 0 123008 0 1764 0 1755 0 0 0 168550 0 165 0 0 0 1952 0 262407 0 1609 0 266133 0 87.13 0 1997910 0 41354 268156 6.484402959810 2292987.0 2120918.0 46524.0 123008.0 1764.0 1755.0 0.0 168550.0 1997910.0 92.5 2.0 5.4 0.1 0.1 0.0 7.4 87.1 50 50 50.00 38 114649350 26.9 22.2 22.3 28.6 0.0 37.2 24.0 smartseq 1444628 SRR4251058 SRP090061 SRS1698736 SRX2171059 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317277: 26Dp4_E02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317277 GSM2317277 26Dp4_E02_smart-seq 210332100 2103321 2016-09-30 15:56:31 147230798 210332100 2103321 2 2103321 index:0,count:2103321,average:50,stdev:0|index:1,count:2103321,average:50,stdev:0 GSM2317277_r1 3.6 3.23 0.07 185231542 236264154 175421139 225162091 127.55 128.36 1943133 1588316 266.946 2494.143 111 5844 70.67 74.74 2209957 1373294 2209957 1373294 71.23 71.75 2209957 1384077 2209957 1318419 28368482 15.32 1.89 0 5.02 0 0.07 0 0.10 0 0.00 0 7.44 0 1943133 0 100 0 98.70 0 1.37 0 0.01 0 1.20 0 0.01 0 280.44 0 0.27 0 39794 0 2103321 0 105690 0 1524 0 2176 0 0 0 156488 0 153 0 0 0 1729 0 199693 0 1565 0 203140 0 87.36 0 1837443 0 35961 207069 5.758154667557 2103321.0 1943133.0 39794.0 105690.0 1524.0 2176.0 0.0 156488.0 1837443.0 92.4 1.9 5.0 0.1 0.1 0.0 7.4 87.4 50 50 50.00 38 105166050 27.1 22.1 22.3 28.4 0.0 37.1 23.5 smartseq 1444629 SRR4252058 SRP090061 SRS1699734 SRX2172059 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318277: SK_2_H09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318277 GSM2318277 SK_2_H09_smart-seq 211849100 2118491 2016-09-30 15:56:31 147007527 211849100 2118491 2 2118491 index:0,count:2118491,average:50,stdev:0|index:1,count:2118491,average:50,stdev:0 GSM2318277_r1 5.13 2.53 0.07 182073463 237922199 169764235 224548826 130.67 132.27 1955328 1600541 224.947 1880.171 78 7415 76.8 82.56 2337553 1501669 2337553 1501669 77.51 79.01 2337553 1515511 2337553 1437035 17369664 9.54 1.99 0 6.44 0 0.09 0 0.07 0 0.00 0 7.54 0 1955328 0 100 0 98.48 0 1.38 0 0.01 0 1.19 0 0.01 0 254.22 0 0.23 0 42104 0 2118491 0 136533 0 2009 0 1492 0 0 0 159662 0 175 0 0 0 1882 0 236429 0 1493 0 239979 0 85.85 0 1818795 0 42673 241530 5.660019215898 2118491.0 1955328.0 42104.0 136533.0 2009.0 1492.0 0.0 159662.0 1818795.0 92.3 2.0 6.4 0.1 0.1 0.0 7.5 85.9 50 50 50.00 38 105924550 27.1 22.2 22.3 28.5 0.0 37.3 24.3 smartseq 1444661 SRR4251059 SRP090061 SRS1698735 SRX2171060 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317278: 26Dp4_E03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317278 GSM2317278 26Dp4_E03_smart-seq 290929600 2909296 2016-09-30 15:56:31 203407569 290929600 2909296 2 2909296 index:0,count:2909296,average:50,stdev:0|index:1,count:2909296,average:50,stdev:0 GSM2317278_r1 0.07 3.46 0.13 255898489 313610017 244838702 301949617 122.55 123.33 2678885 2230035 259.329 2438.342 110 8143 66.47 69.54 2988362 1780549 2988362 1780549 66.11 66.59 2988362 1770907 2988362 1705092 47319673 18.49 1.66 0 4.06 0 0.09 0 0.11 0 0.00 0 7.72 0 2678885 0 100 0 98.80 0 1.43 0 0.01 0 1.21 0 0.01 0 361.15 0 0.26 0 48397 0 2909296 0 118241 0 2481 0 3306 0 0 0 224624 0 173 0 0 0 2093 0 250406 0 2170 0 254842 0 88.02 0 2560644 0 33015 262797 7.959927305770 2909296.0 2678885.0 48397.0 118241.0 2481.0 3306.0 0.0 224624.0 2560644.0 92.1 1.7 4.1 0.1 0.1 0.0 7.7 88.0 50 50 50.00 38 145464800 27.4 21.8 21.9 28.9 0.0 37.2 23.7 smartseq 1444662 SRR4252059 SRP090061 SRS1699736 SRX2172060 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318278: SK_2_H10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318278 GSM2318278 SK_2_H10_smart-seq 196156400 1961564 2016-09-30 15:56:31 137049291 196156400 1961564 2 1961564 index:0,count:1961564,average:50,stdev:0|index:1,count:1961564,average:50,stdev:0 GSM2318278_r1 5.43 2.65 0.11 150374843 199512307 133987133 181835108 132.68 135.71 1661124 1450000 185.873 1257.192 83 8838 86.61 97.53 2231310 1438636 2231310 1438636 89.73 93.13 2231310 1490587 2231310 1373808 1519340 1.01 2.58 0 9.48 0 0.07 0 0.01 0 0.00 0 15.24 0 1661124 0 100 0 97.97 0 1.31 0 0.01 0 1.16 0 0.01 0 243.50 0 0.22 0 50589 0 1961564 0 186019 0 1336 0 230 0 0 0 298874 0 177 0 0 0 1325 0 189456 0 758 0 191716 0 75.20 0 1475105 0 17896 192880 10.777827447474 1961564.0 1661124.0 50589.0 186019.0 1336.0 230.0 0.0 298874.0 1475105.0 84.7 2.6 9.5 0.1 0.0 0.0 15.2 75.2 50 50 50.00 38 98078200 25.6 22.9 23.0 28.6 0.0 37.0 21.8 smartseq 1444882 SRR4251060 SRP090061 SRS1698737 SRX2171061 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317279: 26Dp4_E04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317279 GSM2317279 26Dp4_E04_smart-seq 172228000 1722280 2016-09-30 15:56:31 121161769 172228000 1722280 2 1722280 index:0,count:1722280,average:50,stdev:0|index:1,count:1722280,average:50,stdev:0 GSM2317279_r1 4.57 3.19 0.2 150113035 189097289 143064235 181920815 125.97 127.16 1591490 1332204 261.051 2489.631 80 4970 65.65 69.01 1806968 1044751 1806968 1044751 65.44 66.09 1806968 1041448 1806968 1000623 28166962 18.76 1.99 0 4.50 0 0.13 0 0.12 0 0.00 0 7.35 0 1591490 0 100 0 98.68 0 1.35 0 0.01 0 1.22 0 0.01 0 221.44 0 0.29 0 34290 0 1722280 0 77496 0 2160 0 2096 0 0 0 126534 0 118 0 0 0 1231 0 143566 0 1289 0 146204 0 87.91 0 1513994 0 38208 147584 3.862646566164 1722280.0 1591490.0 34290.0 77496.0 2160.0 2096.0 0.0 126534.0 1513994.0 92.4 2.0 4.5 0.1 0.1 0.0 7.3 87.9 50 50 50.00 38 86114000 27.2 22.0 22.1 28.7 0.0 37.0 23.4 smartseq 1444883 SRR4252060 SRP090061 SRS1699735 SRX2172061 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318279: SK_2_H11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318279 GSM2318279 SK_2_H11_smart-seq 80856400 808564 2016-09-30 15:56:31 56275617 80856400 808564 2 808564 index:0,count:808564,average:50,stdev:0|index:1,count:808564,average:50,stdev:0 GSM2318279_r1 0.01 0.18 0.01 39167760 38959656 39001950 38850491 99.47 99.61 445324 443098 148.254 672.878 105 3357 97.9 98.49 453387 435966 453387 435966 97.96 98.48 453387 436232 453387 435942 248964 0.64 1.23 0 0.33 0 0.01 0 0.00 0 0.00 0 44.92 0 445324 0 100 0 97.66 0 1.51 0 0.01 0 1.19 0 0.01 0 171.23 0 0.17 0 9950 0 808564 0 2653 0 46 0 12 0 0 0 363182 0 0 0 0 0 1 0 103 0 40 0 144 0 54.75 0 442671 0 81 81 1.000000000000 808564.0 445324.0 9950.0 2653.0 46.0 12.0 0.0 363182.0 442671.0 55.1 1.2 0.3 0.0 0.0 0.0 44.9 54.7 50 50 50.00 38 40428200 24.6 20.5 19.6 35.3 0.0 35.5 16.2 smartseq 1444914 SRR4251061 SRP090061 SRS1698739 SRX2171062 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317280: 26Dp4_E05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317280 GSM2317280 26Dp4_E05_smart-seq 318968800 3189688 2016-09-30 15:56:31 221612023 318968800 3189688 2 3189688 index:0,count:3189688,average:50,stdev:0|index:1,count:3189688,average:50,stdev:0 GSM2317280_r1 3.22 3.22 0.16 280411995 350780238 267165743 336399601 125.09 125.91 2969340 2489990 248.909 2432.052 110 9714 66.64 70.05 3344384 1978755 3344384 1978755 66.75 67.17 3344384 1981956 3344384 1897360 51159165 18.24 1.78 0 4.54 0 0.07 0 0.13 0 0.00 0 6.71 0 2969340 0 100 0 98.76 0 1.36 0 0.01 0 1.21 0 0.01 0 328.08 0 0.25 0 56805 0 3189688 0 144697 0 2369 0 4076 0 0 0 213903 0 176 0 0 0 2355 0 282832 0 2311 0 287674 0 88.56 0 2824643 0 39110 293675 7.508949117873 3189688.0 2969340.0 56805.0 144697.0 2369.0 4076.0 0.0 213903.0 2824643.0 93.1 1.8 4.5 0.1 0.1 0.0 6.7 88.6 50 50 50.00 38 159484400 27.3 21.9 22.0 28.7 0.0 37.2 23.8 smartseq 1444915 SRR4252061 SRP090061 SRS1699742 SRX2172062 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318280: SK_2_H12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318280 GSM2318280 SK_2_H12_smart-seq 291786300 2917863 2016-09-30 15:56:31 202667253 291786300 2917863 2 2917863 index:0,count:2917863,average:50,stdev:0|index:1,count:2917863,average:50,stdev:0 GSM2318280_r1 5.49 3.12 0.12 242519411 311168645 225901781 292816329 128.31 129.62 2640103 2255933 204.538 1696.711 78 11278 71.11 76.57 3145019 1877400 3145019 1877400 72.56 73.37 3145019 1915731 3145019 1798982 33212671 13.69 2.16 0 6.45 0 0.09 0 0.10 0 0.00 0 9.32 0 2640103 0 100 0 98.36 0 1.34 0 0.01 0 1.19 0 0.01 0 375.15 0 0.23 0 62886 0 2917863 0 188231 0 2737 0 2946 0 0 0 272077 0 257 0 0 0 1973 0 271056 0 2115 0 275401 0 84.03 0 2451872 0 33224 278661 8.387340476764 2917863.0 2640103.0 62886.0 188231.0 2737.0 2946.0 0.0 272077.0 2451872.0 90.5 2.2 6.5 0.1 0.1 0.0 9.3 84.0 50 50 50.00 38 145893150 27.1 21.8 21.9 29.2 0.0 37.2 23.2 smartseq 1444945 SRR4251062 SRP090061 SRS1698738 SRX2171063 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317281: 26Dp4_E06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317281 GSM2317281 26Dp4_E06_smart-seq 172963000 1729630 2016-09-30 15:56:31 121065869 172963000 1729630 2 1729630 index:0,count:1729630,average:50,stdev:0|index:1,count:1729630,average:50,stdev:0 GSM2317281_r1 1.77 3.19 0.18 150316565 186127445 142596893 177989466 123.82 124.82 1592507 1328249 261.717 2478.491 81 4989 65.5 69.16 1824043 1043069 1824043 1043069 65.6 66.15 1824043 1044697 1824043 997582 27462143 18.27 1.89 0 4.88 0 0.09 0 0.14 0 0.00 0 7.70 0 1592507 0 100 0 98.69 0 1.41 0 0.01 0 1.22 0 0.01 0 222.38 0 0.27 0 32614 0 1729630 0 84372 0 1517 0 2440 0 0 0 133166 0 114 0 0 0 1296 0 150062 0 1259 0 152731 0 87.19 0 1508135 0 32835 156273 4.759342165372 1729630.0 1592507.0 32614.0 84372.0 1517.0 2440.0 0.0 133166.0 1508135.0 92.1 1.9 4.9 0.1 0.1 0.0 7.7 87.2 50 50 50.00 38 86481500 27.2 22.0 22.1 28.7 0.0 37.1 23.4 smartseq 1444946 SRR4252062 SRP090061 SRS1699738 SRX2172063 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318281: SK_3_A01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318281 GSM2318281 SK_3_A01_smart-seq 184052100 1840521 2016-09-30 15:56:31 127616500 184052100 1840521 2 1840521 index:0,count:1840521,average:50,stdev:0|index:1,count:1840521,average:50,stdev:0 GSM2318281_r1 15.44 1.76 0.07 134424178 189059125 114727068 165837619 140.64 144.55 1511523 1399499 176.518 815.636 51 9217 83.69 98.67 2171584 1265026 2171584 1265026 90.66 94.74 2171584 1370320 2171584 1214612 685893 0.51 2.93 0 12.46 0 0.11 0 0.01 0 0.00 0 17.76 0 1511523 0 100 0 97.82 0 1.23 0 0.01 0 1.11 0 0.00 0 194.88 0 0.27 0 53971 0 1840521 0 229419 0 1955 0 130 0 0 0 326913 0 88 0 0 0 477 0 103941 0 493 0 104999 0 69.66 0 1282104 0 4929 107099 21.728342462974 1840521.0 1511523.0 53971.0 229419.0 1955.0 130.0 0.0 326913.0 1282104.0 82.1 2.9 12.5 0.1 0.0 0.0 17.8 69.7 50 50 50.00 38 92026050 25.3 23.0 22.8 28.9 0.0 37.1 21.3 smartseq 1444977 SRR4251063 SRP090061 SRS1698740 SRX2171064 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317282: 26Dp4_E07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317282 GSM2317282 26Dp4_E07_smart-seq 219882500 2198825 2016-09-30 15:56:31 154186994 219882500 2198825 2 2198825 index:0,count:2198825,average:50,stdev:0|index:1,count:2198825,average:50,stdev:0 GSM2317282_r1 2.07 3.17 0.15 194410397 249246387 182303694 235928478 128.21 129.42 2033332 1652620 273.985 2443.352 105 5714 71.82 76.7 2371933 1460242 2371933 1460242 72.41 73.13 2371933 1472256 2371933 1392380 26078517 13.41 1.95 0 5.89 0 0.06 0 0.10 0 0.00 0 7.36 0 2033332 0 100 0 98.70 0 1.33 0 0.01 0 1.25 0 0.01 0 272.96 0 0.27 0 42971 0 2198825 0 129410 0 1356 0 2231 0 0 0 161906 0 162 0 0 0 1668 0 213268 0 1582 0 216680 0 86.59 0 1903922 0 35586 223377 6.277103355252 2198825.0 2033332.0 42971.0 129410.0 1356.0 2231.0 0.0 161906.0 1903922.0 92.5 2.0 5.9 0.1 0.1 0.0 7.4 86.6 50 50 50.00 38 109941250 26.8 22.4 22.6 28.2 0.0 37.0 23.4 smartseq 1444978 SRR4252063 SRP090061 SRS1699739 SRX2172064 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318282: SK_3_A02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318282 GSM2318282 SK_3_A02_smart-seq 137417500 1374175 2016-09-30 15:56:31 96391878 137417500 1374175 2 1374175 index:0,count:1374175,average:50,stdev:0|index:1,count:1374175,average:50,stdev:0 GSM2318282_r1 3.42 3.18 0.09 118392168 155754764 110924774 147546140 131.56 133.01 1263000 971536 254.779 2797.617 80 4027 77.24 82.61 1494325 975483 1494325 975483 77.6 78.84 1494325 980047 1494325 930919 11478951 9.70 2.03 0 5.98 0 0.09 0 0.09 0 0.00 0 7.91 0 1263000 0 100 0 98.43 0 1.37 0 0.01 0 1.18 0 0.01 0 235.57 0 0.28 0 27960 0 1374175 0 82232 0 1239 0 1210 0 0 0 108726 0 88 0 0 0 1192 0 166874 0 1019 0 169173 0 85.93 0 1180768 0 45211 170247 3.765610139125 1374175.0 1263000.0 27960.0 82232.0 1239.0 1210.0 0.0 108726.0 1180768.0 91.9 2.0 6.0 0.1 0.1 0.0 7.9 85.9 50 50 50.00 38 68708750 26.5 22.9 23.0 27.7 0.0 37.3 24.5 smartseq 1445010 SRR4251064 SRP090061 SRS1698741 SRX2171065 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317283: 26Dp4_E08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317283 GSM2317283 26Dp4_E08_smart-seq 156770600 1567706 2016-09-30 15:56:31 109456132 156770600 1567706 2 1567706 index:0,count:1567706,average:50,stdev:0|index:1,count:1567706,average:50,stdev:0 GSM2317283_r1 1.43 3.43 0.19 136504441 163224162 131164580 158019754 119.57 120.47 1425085 1210273 272.077 2471.144 110 4429 59.06 61.54 1576171 841688 1576171 841688 58.86 59.29 1576171 838774 1576171 810950 32225559 23.61 1.76 0 3.65 0 0.10 0 0.18 0 0.00 0 8.81 0 1425085 0 100 0 98.69 0 1.45 0 0.01 0 1.19 0 0.01 0 201.56 0 0.27 0 27662 0 1567706 0 57297 0 1625 0 2877 0 0 0 138119 0 92 0 0 0 1096 0 110384 0 1558 0 113130 0 87.25 0 1367788 0 25871 114903 4.441382242666 1567706.0 1425085.0 27662.0 57297.0 1625.0 2877.0 0.0 138119.0 1367788.0 90.9 1.8 3.7 0.1 0.2 0.0 8.8 87.2 50 50 50.00 38 78385300 27.9 21.2 21.5 29.3 0.0 37.2 23.8 smartseq 1445011 SRR4252064 SRP090061 SRS1699740 SRX2172065 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318283: SK_3_A03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318283 GSM2318283 SK_3_A03_smart-seq 271038000 2710380 2016-09-30 15:56:31 188051965 271038000 2710380 2 2710380 index:0,count:2710380,average:50,stdev:0|index:1,count:2710380,average:50,stdev:0 GSM2318283_r1 5.08 2.67 0.13 228027601 297099728 205616404 275533026 130.29 134.0 2476663 2039827 209.671 1935.923 83 10579 75.68 84.17 3341474 1874265 3341474 1874265 77.67 80.25 3341474 1923589 3341474 1786972 17811948 7.81 2.20 0 9.22 0 0.12 0 0.07 0 0.00 0 8.44 0 2476663 0 100 0 98.31 0 1.30 0 0.01 0 1.18 0 0.01 0 304.92 0 0.26 0 59677 0 2710380 0 249789 0 3165 0 1829 0 0 0 228723 0 184 0 0 0 2302 0 303386 0 1721 0 307593 0 82.16 0 2226874 0 38262 311441 8.139694736292 2710380.0 2476663.0 59677.0 249789.0 3165.0 1829.0 0.0 228723.0 2226874.0 91.4 2.2 9.2 0.1 0.1 0.0 8.4 82.2 50 50 50.00 38 135519000 26.3 23.0 23.1 27.6 0.0 37.5 24.8 smartseq 1445043 SRR4251065 SRP090061 SRS1698742 SRX2171066 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317284: 26Dp4_E09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317284 GSM2317284 26Dp4_E09_smart-seq 149140300 1491403 2016-09-30 15:56:31 103644423 149140300 1491403 2 1491403 index:0,count:1491403,average:50,stdev:0|index:1,count:1491403,average:50,stdev:0 GSM2317284_r1 6.54 3.05 0.09 128015509 172557604 119767435 163430595 134.79 136.46 1356708 1059178 247.941 2688.432 108 4240 90.52 96.97 1620073 1228043 1620073 1228043 90.31 92.27 1620073 1225271 1620073 1168535 1285778 1.00 2.03 0 6.06 0 0.05 0 0.01 0 0.00 0 8.97 0 1356708 0 100 0 98.17 0 1.29 0 0.01 0 1.18 0 0.01 0 268.45 0 0.26 0 30337 0 1491403 0 90328 0 763 0 175 0 0 0 133757 0 178 0 0 0 1491 0 174768 0 726 0 177163 0 84.91 0 1266380 0 24088 181394 7.530471604118 1491403.0 1356708.0 30337.0 90328.0 763.0 175.0 0.0 133757.0 1266380.0 91.0 2.0 6.1 0.1 0.0 0.0 9.0 84.9 50 50 50.00 38 74570150 26.3 22.9 23.2 27.6 0.0 37.2 23.6 smartseq 1445044 SRR4252065 SRP090061 SRS1699743 SRX2172066 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318284: SK_3_A04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318284 GSM2318284 SK_3_A04_smart-seq 142101000 1421010 2016-09-30 15:56:31 98187085 142101000 1421010 2 1421010 index:0,count:1421010,average:50,stdev:0|index:1,count:1421010,average:50,stdev:0 GSM2318284_r1 13.2 0.99 0.09 102874512 133738531 89065209 120507241 130.0 135.3 1157615 1094622 167.967 640.416 73 7007 85.35 99.03 1752707 988072 1752707 988072 91.02 96.74 1752707 1053719 1752707 965231 367114 0.36 2.58 0 11.25 0 0.11 0 0.00 0 0.00 0 18.42 0 1157615 0 100 0 97.77 0 1.25 0 0.01 0 1.12 0 0.01 0 426.30 0 0.26 0 36684 0 1421010 0 159836 0 1517 0 63 0 0 0 261815 0 30 0 0 0 215 0 59164 0 341 0 59750 0 70.22 0 997779 0 3283 59920 18.251599147122 1421010.0 1157615.0 36684.0 159836.0 1517.0 63.0 0.0 261815.0 997779.0 81.5 2.6 11.2 0.1 0.0 0.0 18.4 70.2 50 50 50.00 38 71050500 25.6 22.7 22.7 29.0 0.0 37.1 21.4 smartseq 1445074 SRR4251066 SRP090061 SRS1698743 SRX2171067 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317285: 26Dp4_E10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317285 GSM2317285 26Dp4_E10_smart-seq 173811700 1738117 2016-09-30 15:56:31 120759630 173811700 1738117 2 1738117 index:0,count:1738117,average:50,stdev:0|index:1,count:1738117,average:50,stdev:0 GSM2317285_r1 11.26 2.65 0.14 142388824 193181365 130533681 179932873 135.67 137.84 1524560 1304651 217.457 1823.367 105 6216 89.9 98.35 1899831 1370635 1899831 1370635 92.09 94.81 1899831 1403974 1899831 1321369 583542 0.41 2.08 0 7.53 0 0.03 0 0.01 0 0.00 0 12.25 0 1524560 0 100 0 98.12 0 1.24 0 0.01 0 1.15 0 0.01 0 240.66 0 0.25 0 36146 0 1738117 0 130882 0 537 0 89 0 0 0 212931 0 110 0 0 0 955 0 149556 0 868 0 151489 0 80.18 0 1393678 0 13323 155130 11.643773924792 1738117.0 1524560.0 36146.0 130882.0 537.0 89.0 0.0 212931.0 1393678.0 87.7 2.1 7.5 0.0 0.0 0.0 12.3 80.2 50 50 50.00 38 86905850 26.2 22.9 23.2 27.6 0.0 37.2 23.3 smartseq 1445075 SRR4252066 SRP090061 SRS1699741 SRX2172067 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318285: SK_3_A05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318285 GSM2318285 SK_3_A05_smart-seq 178644500 1786445 2016-09-30 15:56:31 124038324 178644500 1786445 2 1786445 index:0,count:1786445,average:50,stdev:0|index:1,count:1786445,average:50,stdev:0 GSM2318285_r1 4.88 3.23 0.1 149671860 191972755 137701939 180385166 128.26 131.0 1604861 1330988 237.317 2141.430 83 5707 71.16 77.54 2032198 1142049 2032198 1142049 72.73 74.16 2032198 1167282 2032198 1092349 17959244 12.00 2.02 0 7.39 0 0.10 0 0.10 0 0.00 0 9.97 0 1604861 0 100 0 98.45 0 1.33 0 0.01 0 1.20 0 0.01 0 214.37 0 0.27 0 36083 0 1786445 0 131996 0 1767 0 1711 0 0 0 178106 0 118 0 0 0 1287 0 165138 0 1171 0 167714 0 82.45 0 1472865 0 36504 169463 4.642313171159 1786445.0 1604861.0 36083.0 131996.0 1767.0 1711.0 0.0 178106.0 1472865.0 89.8 2.0 7.4 0.1 0.1 0.0 10.0 82.4 50 50 50.00 38 89322250 26.6 22.5 22.7 28.2 0.0 37.3 23.9 smartseq 1445106 SRR4251067 SRP090061 SRS1698745 SRX2171068 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317286: 26Dp4_E11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317286 GSM2317286 26Dp4_E11_smart-seq 276043000 2760430 2016-09-30 15:56:31 191590155 276043000 2760430 2 2760430 index:0,count:2760430,average:50,stdev:0|index:1,count:2760430,average:50,stdev:0 GSM2317286_r1 1.27 2.82 0.16 237245995 293068061 226104416 281403555 123.53 124.46 2527552 2130011 243.539 2366.542 81 8500 65.32 68.65 2866772 1650993 2866772 1650993 65.04 65.56 2866772 1643964 2866772 1576732 45413384 19.14 1.87 0 4.44 0 0.07 0 0.12 0 0.00 0 8.25 0 2527552 0 100 0 98.66 0 1.39 0 0.01 0 1.21 0 0.01 0 368.06 0 0.25 0 51591 0 2760430 0 122522 0 1971 0 3186 0 0 0 227721 0 187 0 0 0 1913 0 233977 0 2037 0 238114 0 87.13 0 2405030 0 33104 242741 7.332678830353 2760430.0 2527552.0 51591.0 122522.0 1971.0 3186.0 0.0 227721.0 2405030.0 91.6 1.9 4.4 0.1 0.1 0.0 8.2 87.1 50 50 50.00 38 138021500 27.4 21.8 21.9 28.9 0.0 37.2 23.8 smartseq 1445107 SRR4252067 SRP090061 SRS1699744 SRX2172068 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318286: SK_3_A06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318286 GSM2318286 SK_3_A06_smart-seq 213631100 2136311 2016-09-30 15:56:31 147993936 213631100 2136311 2 2136311 index:0,count:2136311,average:50,stdev:0|index:1,count:2136311,average:50,stdev:0 GSM2318286_r1 5.4 2.89 0.11 182443434 234202627 169587867 220761677 128.37 130.18 1965645 1666984 218.297 2067.750 81 7928 68.98 74.38 2404127 1355876 2404127 1355876 70.02 71.17 2404127 1376359 2404127 1297493 25738139 14.11 1.90 0 6.68 0 0.12 0 0.11 0 0.00 0 7.76 0 1965645 0 100 0 98.51 0 1.32 0 0.01 0 1.18 0 0.01 0 219.73 0 0.25 0 40570 0 2136311 0 142638 0 2559 0 2369 0 0 0 165738 0 148 0 0 0 1593 0 188087 0 1730 0 191558 0 85.33 0 1823007 0 35051 196300 5.600410829934 2136311.0 1965645.0 40570.0 142638.0 2559.0 2369.0 0.0 165738.0 1823007.0 92.0 1.9 6.7 0.1 0.1 0.0 7.8 85.3 50 50 50.00 38 106815550 26.9 22.3 22.4 28.4 0.0 37.4 24.2 smartseq 1445139 SRR4251068 SRP090061 SRS1698744 SRX2171069 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317287: 26Dp4_F01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317287 GSM2317287 26Dp4_F01_smart-seq 254589500 2545895 2016-09-30 15:56:31 176387355 254589500 2545895 2 2545895 index:0,count:2545895,average:50,stdev:0|index:1,count:2545895,average:50,stdev:0 GSM2317287_r1 1.4 3.53 0.21 219829471 267544565 210488051 258206824 121.71 122.67 2339319 2020242 248.046 2209.700 80 7817 60.52 63.3 2624892 1415822 2624892 1415822 60.24 60.67 2624892 1409092 2624892 1357035 49471711 22.50 1.84 0 4.03 0 0.09 0 0.16 0 0.00 0 7.86 0 2339319 0 100 0 98.70 0 1.41 0 0.01 0 1.22 0 0.01 0 398.49 0 0.26 0 46865 0 2545895 0 102723 0 2307 0 4182 0 0 0 200087 0 102 0 0 0 1527 0 182606 0 1970 0 186205 0 87.85 0 2236596 0 25375 189087 7.451704433498 2545895.0 2339319.0 46865.0 102723.0 2307.0 4182.0 0.0 200087.0 2236596.0 91.9 1.8 4.0 0.1 0.2 0.0 7.9 87.9 50 50 50.00 38 127294750 27.7 21.3 21.4 29.5 0.0 37.3 23.7 smartseq 1445140 SRR4252068 SRP090061 SRS1699745 SRX2172069 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318287: SK_3_A07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318287 GSM2318287 SK_3_A07_smart-seq 214336000 2143360 2016-09-30 15:56:31 149279153 214336000 2143360 2 2143360 index:0,count:2143360,average:50,stdev:0|index:1,count:2143360,average:50,stdev:0 GSM2318287_r1 7.27 2.57 0.06 184700176 248356913 169931947 231442224 134.46 136.2 1975715 1552713 238.933 2349.373 81 6853 78.23 85.25 2426692 1545611 2426692 1545611 80.02 81.59 2426692 1580951 2426692 1479334 15671634 8.48 2.08 0 7.59 0 0.08 0 0.06 0 0.00 0 7.69 0 1975715 0 100 0 98.42 0 1.31 0 0.01 0 1.18 0 0.01 0 296.77 0 0.27 0 44636 0 2143360 0 162676 0 1748 0 1179 0 0 0 164718 0 172 0 0 0 1882 0 257312 0 1360 0 260726 0 84.59 0 1813039 0 46241 264831 5.727190155922 2143360.0 1975715.0 44636.0 162676.0 1748.0 1179.0 0.0 164718.0 1813039.0 92.2 2.1 7.6 0.1 0.1 0.0 7.7 84.6 50 50 50.00 38 107168000 26.5 22.8 23.0 27.6 0.0 37.3 24.6 smartseq 1445171 SRR4251069 SRP090061 SRS1698747 SRX2171070 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317288: 26Dp4_F02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317288 GSM2317288 26Dp4_F02_smart-seq 217540300 2175403 2016-09-30 15:56:31 151568205 217540300 2175403 2 2175403 index:0,count:2175403,average:50,stdev:0|index:1,count:2175403,average:50,stdev:0 GSM2317288_r1 2.73 3.49 0.17 190493894 241380577 181055009 231075506 126.71 127.63 2009850 1656161 259.430 2565.613 103 6199 70.45 74.23 2276226 1415884 2276226 1415884 70.38 70.98 2276226 1414471 2276226 1353894 28736976 15.09 1.82 0 4.71 0 0.10 0 0.12 0 0.00 0 7.38 0 2009850 0 100 0 98.68 0 1.38 0 0.01 0 1.24 0 0.01 0 290.05 0 0.27 0 39503 0 2175403 0 102361 0 2266 0 2675 0 0 0 160612 0 136 0 0 0 1744 0 201663 0 1510 0 205053 0 87.68 0 1907489 0 32835 211356 6.436911831887 2175403.0 2009850.0 39503.0 102361.0 2266.0 2675.0 0.0 160612.0 1907489.0 92.4 1.8 4.7 0.1 0.1 0.0 7.4 87.7 50 50 50.00 38 108770150 27.2 22.0 22.1 28.7 0.0 37.2 23.6 smartseq 1445172 SRR4252069 SRP090061 SRS1699746 SRX2172070 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318288: SK_3_A08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318288 GSM2318288 SK_3_A08_smart-seq 209829400 2098294 2016-09-30 15:56:31 145864588 209829400 2098294 2 2098294 index:0,count:2098294,average:50,stdev:0|index:1,count:2098294,average:50,stdev:0 GSM2318288_r1 5.67 3.06 0.08 179381480 231123878 164851918 216415686 128.84 131.28 1929296 1595041 229.763 2116.696 80 7069 70.1 76.48 2433322 1352494 2433322 1352494 71.75 73.04 2433322 1384267 2433322 1291667 23160766 12.91 2.10 0 7.66 0 0.13 0 0.11 0 0.00 0 7.81 0 1929296 0 100 0 98.45 0 1.34 0 0.01 0 1.19 0 0.01 0 236.06 0 0.27 0 44163 0 2098294 0 160789 0 2754 0 2399 0 0 0 163845 0 222 0 0 0 1474 0 204170 0 1464 0 207330 0 84.28 0 1768507 0 38395 209339 5.452246386248 2098294.0 1929296.0 44163.0 160789.0 2754.0 2399.0 0.0 163845.0 1768507.0 91.9 2.1 7.7 0.1 0.1 0.0 7.8 84.3 50 50 50.00 38 104914700 26.6 22.6 22.8 28.0 0.0 37.4 24.5 smartseq 1445393 SRR4251070 SRP090061 SRS1698746 SRX2171071 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317289: 26Dp4_F03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317289 GSM2317289 26Dp4_F03_smart-seq 250680100 2506801 2016-09-30 15:56:31 175042213 250680100 2506801 2 2506801 index:0,count:2506801,average:50,stdev:0|index:1,count:2506801,average:50,stdev:0 GSM2317289_r1 0.39 2.8 0.12 216848904 262094488 207599188 252649047 120.87 121.7 2299463 1940057 256.545 2329.247 100 7360 62.86 65.75 2561055 1445460 2561055 1445460 62.73 63.13 2561055 1442524 2561055 1387894 44685374 20.61 1.79 0 4.03 0 0.10 0 0.14 0 0.00 0 8.03 0 2299463 0 100 0 98.73 0 1.45 0 0.01 0 1.22 0 0.01 0 311.19 0 0.26 0 44856 0 2506801 0 100955 0 2594 0 3395 0 0 0 201349 0 133 0 0 0 1580 0 200710 0 1907 0 204330 0 87.70 0 2198508 0 32158 210806 6.555320604515 2506801.0 2299463.0 44856.0 100955.0 2594.0 3395.0 0.0 201349.0 2198508.0 91.7 1.8 4.0 0.1 0.1 0.0 8.0 87.7 50 50 50.00 38 125340050 27.6 21.5 21.6 29.3 0.0 37.2 23.7 smartseq 1445394 SRR4252070 SRP090061 SRS1699747 SRX2172071 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318289: SK_3_A09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318289 GSM2318289 SK_3_A09_smart-seq 168535700 1685357 2016-09-30 15:56:31 115532662 168535700 1685357 2 1685357 index:0,count:1685357,average:50,stdev:0|index:1,count:1685357,average:50,stdev:0 GSM2318289_r1 2.83 0.75 0.03 138336045 140451351 91654473 115957340 101.53 126.52 1572362 1452765 155.912 1220.398 64 10467 42.29 63.84 4708841 664913 4708841 664913 48.65 59.5 4708841 764980 4708841 619684 20689605 14.96 2.23 0 31.50 0 0.32 0 0.26 0 0.00 0 6.13 0 1572362 0 100 0 97.57 0 1.25 0 0.01 0 1.18 0 0.01 0 224.71 0 0.26 0 37589 0 1685357 0 530872 0 5312 0 4365 0 0 0 103318 0 73 0 0 0 902 0 108643 0 497 0 110115 0 61.80 0 1041490 0 9062 110587 12.203376738027 1685357.0 1572362.0 37589.0 530872.0 5312.0 4365.0 0.0 103318.0 1041490.0 93.3 2.2 31.5 0.3 0.3 0.0 6.1 61.8 50 50 50.00 38 84267850 25.1 24.4 24.7 25.7 0.0 37.5 26.5 smartseq 1445424 SRR4252071 SRP090061 SRS1699748 SRX2172072 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318290: SK_3_A10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318290 GSM2318290 SK_3_A10_smart-seq 197429400 1974294 2016-09-30 15:56:31 138004871 197429400 1974294 2 1974294 index:0,count:1974294,average:50,stdev:0|index:1,count:1974294,average:50,stdev:0 GSM2318290_r1 5.62 3.36 0.08 169712747 228782428 155801514 213895301 134.81 137.29 1816431 1421188 239.470 2563.479 81 6249 78.95 86.22 2270407 1434054 2270407 1434054 80.37 82.1 2270407 1459914 2270407 1365478 12420710 7.32 2.14 0 7.76 0 0.09 0 0.07 0 0.00 0 7.84 0 1816431 0 100 0 98.38 0 1.31 0 0.01 0 1.17 0 0.00 0 229.27 0 0.28 0 42176 0 1974294 0 153244 0 1691 0 1433 0 0 0 154739 0 177 0 0 0 1667 0 244617 0 1352 0 247813 0 84.24 0 1663187 0 44465 251638 5.659237602609 1974294.0 1816431.0 42176.0 153244.0 1691.0 1433.0 0.0 154739.0 1663187.0 92.0 2.1 7.8 0.1 0.1 0.0 7.8 84.2 50 50 50.00 38 98714700 26.0 23.3 23.5 27.2 0.0 37.3 24.5 smartseq 1445456 SRR4251072 SRP090061 SRS1698749 SRX2171073 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317291: 26Dp4_F05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317291 GSM2317291 26Dp4_F05_smart-seq 213661300 2136613 2016-09-30 15:56:31 148821326 213661300 2136613 2 2136613 index:0,count:2136613,average:50,stdev:0|index:1,count:2136613,average:50,stdev:0 GSM2317291_r1 3.51 3.26 0.13 183628157 234780279 172801785 223111401 127.86 129.11 1952054 1594892 262.123 2605.478 81 6079 70.81 75.41 2282841 1382196 2282841 1382196 71.28 72.05 2282841 1391443 2282841 1320617 26381365 14.37 2.05 0 5.57 0 0.08 0 0.09 0 0.00 0 8.47 0 1952054 0 100 0 98.57 0 1.36 0 0.01 0 1.22 0 0.01 0 366.28 0 0.27 0 43862 0 2136613 0 119112 0 1608 0 1948 0 0 0 181003 0 179 0 0 0 1521 0 205124 0 1432 0 208256 0 85.79 0 1832942 0 36982 211757 5.725947758369 2136613.0 1952054.0 43862.0 119112.0 1608.0 1948.0 0.0 181003.0 1832942.0 91.4 2.1 5.6 0.1 0.1 0.0 8.5 85.8 50 50 50.00 38 106830650 27.0 22.2 22.3 28.5 0.0 37.1 23.5 smartseq 1445457 SRR4252072 SRP090061 SRS1699766 SRX2172073 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318291: SK_3_A11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318291 GSM2318291 SK_3_A11_smart-seq 173777200 1737772 2016-09-30 15:56:31 120905043 173777200 1737772 2 1737772 index:0,count:1737772,average:50,stdev:0|index:1,count:1737772,average:50,stdev:0 GSM2318291_r1 4.22 3.53 0.25 144336798 175182885 133811445 165056918 121.37 123.35 1561827 1286458 227.878 2329.037 69 5979 64.12 69.33 1892267 1001481 1892267 1001481 65.42 66.32 1892267 1021727 1892267 957939 21375782 14.81 2.19 0 6.76 0 0.20 0 0.14 0 0.00 0 9.78 0 1561827 0 100 0 98.20 0 1.21 0 0.01 0 1.19 0 0.01 0 250.24 0 0.36 0 37987 0 1737772 0 117387 0 3482 0 2472 0 0 0 169991 0 130 0 0 0 1170 0 156326 0 1180 0 158806 0 83.12 0 1444440 0 39304 160250 4.077193161001 1737772.0 1561827.0 37987.0 117387.0 3482.0 2472.0 0.0 169991.0 1444440.0 89.9 2.2 6.8 0.2 0.1 0.0 9.8 83.1 50 50 50.00 38 86888600 26.8 22.2 22.4 28.6 0.0 37.3 24.0 smartseq 1445488 SRR4251073 SRP090061 SRS1698751 SRX2171074 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317292: 26Dp4_F06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317292 GSM2317292 26Dp4_F06_smart-seq 196114800 1961148 2016-09-30 15:56:31 136984056 196114800 1961148 2 1961148 index:0,count:1961148,average:50,stdev:0|index:1,count:1961148,average:50,stdev:0 GSM2317292_r1 2.8 3.26 0.16 170600809 214279360 161512931 204809174 125.6 126.81 1804914 1490211 265.024 2547.391 90 5492 67.96 71.92 2085584 1226567 2085584 1226567 68.16 68.84 2085584 1230243 2085584 1174119 28286034 16.58 2.01 0 5.07 0 0.07 0 0.12 0 0.00 0 7.77 0 1804914 0 100 0 98.64 0 1.37 0 0.01 0 1.22 0 0.01 0 294.17 0 0.27 0 39429 0 1961148 0 99382 0 1429 0 2447 0 0 0 152358 0 149 0 0 0 1378 0 177352 0 1443 0 180322 0 86.97 0 1705532 0 36874 183172 4.967510983349 1961148.0 1804914.0 39429.0 99382.0 1429.0 2447.0 0.0 152358.0 1705532.0 92.0 2.0 5.1 0.1 0.1 0.0 7.8 87.0 50 50 50.00 38 98057400 27.2 21.9 22.0 28.8 0.0 37.1 23.4 smartseq 1445489 SRR4252073 SRP090061 SRS1699749 SRX2172074 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318292: SK_3_A12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318292 GSM2318292 SK_3_A12_smart-seq 96029600 960296 2016-09-30 15:56:31 66440293 96029600 960296 2 960296 index:0,count:960296,average:50,stdev:0|index:1,count:960296,average:50,stdev:0 GSM2318292_r1 4.64 2.87 0.1 81451208 104107071 76610461 98831270 127.82 129.0 881758 733608 213.190 2110.583 81 3585 73.01 77.8 1026188 643806 1026188 643806 73.7 74.49 1026188 649814 1026188 616380 10270519 12.61 1.99 0 5.65 0 0.10 0 0.11 0 0.00 0 7.97 0 881758 0 100 0 98.46 0 1.32 0 0.01 0 1.18 0 0.01 0 203.36 0 0.25 0 19146 0 960296 0 54269 0 924 0 1063 0 0 0 76551 0 71 0 0 0 703 0 96722 0 615 0 98111 0 86.17 0 827489 0 30261 98428 3.252635405307 960296.0 881758.0 19146.0 54269.0 924.0 1063.0 0.0 76551.0 827489.0 91.8 2.0 5.7 0.1 0.1 0.0 8.0 86.2 50 50 50.00 38 48014800 26.8 22.5 22.6 28.2 0.0 37.5 24.5 smartseq 1445521 SRR4251074 SRP090061 SRS1698752 SRX2171075 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317293: 26Dp4_F08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317293 GSM2317293 26Dp4_F08_smart-seq 229124200 2291242 2016-09-30 15:56:31 159498657 229124200 2291242 2 2291242 index:0,count:2291242,average:50,stdev:0|index:1,count:2291242,average:50,stdev:0 GSM2317293_r1 2.38 3.38 0.13 197029700 246626570 185566523 234624799 125.17 126.44 2098396 1754383 250.434 2414.592 81 6945 67.41 71.72 2452244 1414445 2452244 1414445 67.93 68.58 2452244 1425530 2452244 1352531 33087599 16.79 2.06 0 5.50 0 0.08 0 0.11 0 0.00 0 8.22 0 2098396 0 100 0 98.59 0 1.38 0 0.01 0 1.22 0 0.01 0 343.69 0 0.26 0 47215 0 2291242 0 126107 0 1912 0 2493 0 0 0 188441 0 106 0 0 0 1737 0 201066 0 1494 0 204403 0 86.08 0 1972289 0 32543 208399 6.403804197523 2291242.0 2098396.0 47215.0 126107.0 1912.0 2493.0 0.0 188441.0 1972289.0 91.6 2.1 5.5 0.1 0.1 0.0 8.2 86.1 50 50 50.00 38 114562100 27.1 22.1 22.2 28.7 0.0 37.2 23.6 smartseq 1445522 SRR4252074 SRP090061 SRS1699750 SRX2172075 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318293: SK_3_B01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318293 GSM2318293 SK_3_B01_smart-seq 154150800 1541508 2016-09-30 15:56:31 106603560 154150800 1541508 2 1541508 index:0,count:1541508,average:50,stdev:0|index:1,count:1541508,average:50,stdev:0 GSM2318293_r1 7.59 3.27 0.08 133137742 183672131 122715692 171466772 137.96 139.73 1429450 1128628 239.170 2504.793 81 5164 80.73 87.83 1726862 1153990 1726862 1153990 82.29 83.68 1726862 1176260 1726862 1099477 8835681 6.64 2.25 0 7.50 0 0.07 0 0.06 0 0.00 0 7.14 0 1429450 0 100 0 98.32 0 1.28 0 0.01 0 1.19 0 0.00 0 205.53 0 0.26 0 34613 0 1541508 0 115555 0 1101 0 934 0 0 0 110023 0 143 0 0 0 1333 0 184134 0 994 0 186604 0 85.23 0 1313895 0 39994 188351 4.709481422213 1541508.0 1429450.0 34613.0 115555.0 1101.0 934.0 0.0 110023.0 1313895.0 92.7 2.2 7.5 0.1 0.1 0.0 7.1 85.2 50 50 50.00 38 77075400 26.1 23.3 23.5 27.1 0.0 37.4 24.8 smartseq 1445552 SRR4251075 SRP090061 SRS1698750 SRX2171076 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317294: 26Dp4_F09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317294 GSM2317294 26Dp4_F09_smart-seq 107763000 1077630 2016-09-30 15:56:31 74993170 107763000 1077630 2 1077630 index:0,count:1077630,average:50,stdev:0|index:1,count:1077630,average:50,stdev:0 GSM2317294_r1 3.28 3.19 0.19 94633765 118111435 89533910 112904692 124.81 126.1 992468 818045 281.256 2525.484 111 2647 65.76 69.64 1152829 652666 1152829 652666 66.04 66.71 1152829 655406 1152829 625252 17279129 18.26 1.96 0 5.13 0 0.07 0 0.13 0 0.00 0 7.70 0 992468 0 100 0 98.59 0 1.34 0 0.01 0 1.20 0 0.01 0 193.97 0 0.27 0 21173 0 1077630 0 55264 0 772 0 1384 0 0 0 83006 0 90 0 0 0 882 0 91763 0 840 0 93575 0 86.97 0 937204 0 27996 94548 3.377196742392 1077630.0 992468.0 21173.0 55264.0 772.0 1384.0 0.0 83006.0 937204.0 92.1 2.0 5.1 0.1 0.1 0.0 7.7 87.0 50 50 50.00 38 53881500 27.4 21.8 22.0 28.7 0.0 37.2 23.8 smartseq 1445553 SRR4252075 SRP090061 SRS1699755 SRX2172076 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318294: SK_3_B02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318294 GSM2318294 SK_3_B02_smart-seq 140177000 1401770 2016-09-30 15:56:31 97069621 140177000 1401770 2 1401770 index:0,count:1401770,average:50,stdev:0|index:1,count:1401770,average:50,stdev:0 GSM2318294_r1 5.26 2.74 0.09 121395070 161020328 113449857 151599695 132.64 133.63 1297755 1028419 247.641 2224.562 80 4424 75.65 81.13 1505700 981723 1505700 981723 76.75 77.56 1505700 996034 1505700 938538 11655498 9.60 2.14 0 6.26 0 0.09 0 0.07 0 0.00 0 7.26 0 1297755 0 100 0 98.48 0 1.35 0 0.01 0 1.20 0 0.01 0 219.41 0 0.26 0 29958 0 1401770 0 87722 0 1239 0 958 0 0 0 101818 0 129 0 0 0 1245 0 160271 0 990 0 162635 0 86.32 0 1210033 0 39667 164501 4.147049184461 1401770.0 1297755.0 29958.0 87722.0 1239.0 958.0 0.0 101818.0 1210033.0 92.6 2.1 6.3 0.1 0.1 0.0 7.3 86.3 50 50 50.00 38 70088500 26.5 22.9 22.9 27.7 0.0 37.3 24.5 smartseq 1445584 SRR4251076 SRP090061 SRS1698753 SRX2171077 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317295: 26Dp4_F10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317295 GSM2317295 26Dp4_F10_smart-seq 201101900 2011019 2016-09-30 15:56:31 140497513 201101900 2011019 2 2011019 index:0,count:2011019,average:50,stdev:0|index:1,count:2011019,average:50,stdev:0 GSM2317295_r1 3.55 3.1 0.12 173500389 217277494 164065928 207446733 125.23 126.44 1839763 1512437 259.482 2439.460 81 5787 68.14 72.2 2132543 1253551 2132543 1253551 68.58 69.21 2132543 1261711 2132543 1201719 27948836 16.11 2.01 0 5.15 0 0.10 0 0.14 0 0.00 0 8.28 0 1839763 0 100 0 98.61 0 1.39 0 0.01 0 1.20 0 0.01 0 226.24 0 0.27 0 40356 0 2011019 0 103533 0 1958 0 2760 0 0 0 166538 0 178 0 0 0 1444 0 188334 0 1837 0 191793 0 86.34 0 1736230 0 35449 195591 5.517532229400 2011019.0 1839763.0 40356.0 103533.0 1958.0 2760.0 0.0 166538.0 1736230.0 91.5 2.0 5.1 0.1 0.1 0.0 8.3 86.3 50 50 50.00 38 100550950 27.1 22.0 22.1 28.8 0.0 37.2 23.5 smartseq 1445585 SRR4252076 SRP090061 SRS1699753 SRX2172077 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318295: SK_3_B03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318295 GSM2318295 SK_3_B03_smart-seq 185879100 1858791 2016-09-30 15:56:31 128470738 185879100 1858791 2 1858791 index:0,count:1858791,average:50,stdev:0|index:1,count:1858791,average:50,stdev:0 GSM2318295_r1 5.77 2.58 0.11 155996167 203880273 145420514 192150072 130.7 132.13 1679226 1397845 232.560 1848.994 80 6257 73.47 79.04 2000495 1233772 2000495 1233772 74.61 75.71 2000495 1252892 2000495 1181835 17788713 11.40 2.12 0 6.36 0 0.11 0 0.07 0 0.00 0 9.47 0 1679226 0 100 0 98.50 0 1.35 0 0.01 0 1.21 0 0.01 0 290.94 0 0.25 0 39373 0 1858791 0 118209 0 2085 0 1380 0 0 0 176100 0 150 0 0 0 1346 0 175368 0 1148 0 178012 0 83.98 0 1561017 0 31011 180573 5.822869304440 1858791.0 1679226.0 39373.0 118209.0 2085.0 1380.0 0.0 176100.0 1561017.0 90.3 2.1 6.4 0.1 0.1 0.0 9.5 84.0 50 50 50.00 38 92939550 26.7 22.5 22.5 28.3 0.0 37.4 23.9 smartseq 1445616 SRR4252077 SRP090061 SRS1699752 SRX2172078 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318296: SK_3_B04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318296 GSM2318296 SK_3_B04_smart-seq 59671900 596719 2016-09-30 15:56:31 40952729 59671900 596719 2 596719 index:0,count:596719,average:50,stdev:0|index:1,count:596719,average:50,stdev:0 GSM2318296_r1 0.01 0.15 0.01 34722149 34657325 34671085 34622067 99.81 99.86 390447 389460 162.742 425.843 51 2711 98.94 99.16 392397 386292 392397 386292 98.96 99.15 392397 386369 392397 386257 117935 0.34 1.34 0 0.15 0 0.01 0 0.00 0 0.00 0 34.56 0 390447 0 100 0 97.90 0 1.40 0 0.01 0 1.08 0 0.01 0 113.06 0 0.17 0 8014 0 596719 0 872 0 37 0 12 0 0 0 206223 0 0 0 0 0 0 0 42 0 45 0 87 0 65.29 0 389575 0 29 29 1.000000000000 596719.0 390447.0 8014.0 872.0 37.0 12.0 0.0 206223.0 389575.0 65.4 1.3 0.1 0.0 0.0 0.0 34.6 65.3 50 50 50.00 38 29835950 25.3 21.1 20.7 32.9 0.0 36.4 17.9 smartseq 1445649 SRR4251078 SRP090061 SRS1698757 SRX2171079 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317297: 26Dp4_G01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317297 GSM2317297 26Dp4_G01_smart-seq 200302700 2003027 2016-09-30 15:56:31 139394908 200302700 2003027 2 2003027 index:0,count:2003027,average:50,stdev:0|index:1,count:2003027,average:50,stdev:0 GSM2317297_r1 0.56 3.47 0.2 175791893 213091264 169249663 206438955 121.22 121.97 1836966 1549645 270.531 2484.430 110 5373 61.93 64.38 2021081 1137632 2021081 1137632 61.69 62.08 2021081 1133224 2021081 1096968 38546646 21.93 1.64 0 3.49 0 0.09 0 0.14 0 0.00 0 8.05 0 1836966 0 100 0 98.85 0 1.43 0 0.01 0 1.23 0 0.01 0 300.45 0 0.26 0 32858 0 2003027 0 69873 0 1863 0 2877 0 0 0 161321 0 139 0 0 0 1446 0 151726 0 1502 0 154813 0 88.22 0 1767093 0 30211 158865 5.258515110390 2003027.0 1836966.0 32858.0 69873.0 1863.0 2877.0 0.0 161321.0 1767093.0 91.7 1.6 3.5 0.1 0.1 0.0 8.1 88.2 50 50 50.00 38 100151350 27.9 21.2 21.4 29.5 0.0 37.2 23.6 smartseq 1445650 SRR4252078 SRP090061 SRS1699751 SRX2172079 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318297: SK_3_B05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318297 GSM2318297 SK_3_B05_smart-seq 123548800 1235488 2016-09-30 15:56:31 85630719 123548800 1235488 2 1235488 index:0,count:1235488,average:50,stdev:0|index:1,count:1235488,average:50,stdev:0 GSM2318297_r1 6.11 2.95 0.12 107145487 140162525 100426273 132732546 130.82 132.17 1140043 934335 262.573 2585.862 81 3542 70.84 75.77 1336648 807647 1336648 807647 71.67 72.45 1336648 817102 1336648 772315 14251424 13.30 2.03 0 6.00 0 0.09 0 0.10 0 0.00 0 7.53 0 1140043 0 100 0 98.59 0 1.34 0 0.01 0 1.21 0 0.01 0 247.10 0 0.26 0 25030 0 1235488 0 74078 0 1159 0 1282 0 0 0 93004 0 100 0 0 0 885 0 111402 0 879 0 113266 0 86.28 0 1065965 0 36473 115065 3.154799440682 1235488.0 1140043.0 25030.0 74078.0 1159.0 1282.0 0.0 93004.0 1065965.0 92.3 2.0 6.0 0.1 0.1 0.0 7.5 86.3 50 50 50.00 38 61774400 26.9 22.2 22.3 28.5 0.0 37.3 24.0 smartseq 1445682 SRR4251079 SRP090061 SRS1698756 SRX2171080 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317298: 26Dp4_G02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317298 GSM2317298 26Dp4_G02_smart-seq 249335300 2493353 2016-09-30 15:56:31 172906258 249335300 2493353 2 2493353 index:0,count:2493353,average:50,stdev:0|index:1,count:2493353,average:50,stdev:0 GSM2317298_r1 3.36 2.88 0.08 215687707 283324804 201311848 267888538 131.36 133.07 2276161 1850049 235.236 2461.097 105 7956 89.35 95.87 2746789 2033648 2746789 2033648 89.85 92.09 2746789 2045155 2746789 1953452 3828030 1.77 1.74 0 6.21 0 0.04 0 0.02 0 0.00 0 8.65 0 2276161 0 100 0 98.48 0 1.34 0 0.01 0 1.22 0 0.01 0 332.45 0 0.25 0 43295 0 2493353 0 154826 0 983 0 491 0 0 0 215718 0 202 0 0 0 1912 0 266019 0 980 0 269113 0 85.08 0 2121335 0 16929 276938 16.358792604407 2493353.0 2276161.0 43295.0 154826.0 983.0 491.0 0.0 215718.0 2121335.0 91.3 1.7 6.2 0.0 0.0 0.0 8.7 85.1 50 50 50.00 38 124667650 26.2 22.9 23.1 27.8 0.0 37.2 23.3 smartseq 1445683 SRR4252079 SRP090061 SRS1699756 SRX2172080 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318298: SK_3_B06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318298 GSM2318298 SK_3_B06_smart-seq 141013900 1410139 2016-09-30 15:56:31 97029194 141013900 1410139 2 1410139 index:0,count:1410139,average:50,stdev:0|index:1,count:1410139,average:50,stdev:0 GSM2318298_r1 0.48 2.99 0.13 119637206 148261895 114569555 142823274 123.93 124.66 1287693 1082207 231.714 2203.544 60 4732 71.54 74.83 1434382 921192 1434382 921192 70.9 71.53 1434382 912957 1434382 880636 17241033 14.41 1.88 0 4.01 0 0.08 0 0.09 0 0.00 0 8.51 0 1287693 0 100 0 98.54 0 1.44 0 0.01 0 1.20 0 0.01 0 220.72 0 0.23 0 26543 0 1410139 0 56594 0 1094 0 1313 0 0 0 120039 0 81 0 0 0 1157 0 125376 0 962 0 127576 0 87.30 0 1231099 0 27116 128063 4.722783596401 1410139.0 1287693.0 26543.0 56594.0 1094.0 1313.0 0.0 120039.0 1231099.0 91.3 1.9 4.0 0.1 0.1 0.0 8.5 87.3 50 50 50.00 38 70506950 27.1 22.0 22.0 29.0 0.0 37.4 23.8 smartseq 1445905 SRR4251080 SRP090061 SRS1698755 SRX2171081 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317299: 26Dp4_G03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317299 GSM2317299 26Dp4_G03_smart-seq 251095800 2510958 2016-09-30 15:56:31 174766036 251095800 2510958 2 2510958 index:0,count:2510958,average:50,stdev:0|index:1,count:2510958,average:50,stdev:0 GSM2317299_r1 1.16 3.02 0.07 221778527 267378806 213418932 258585631 120.56 121.16 2320037 1973925 264.531 2456.240 110 7050 59.39 61.78 2554307 1377927 2554307 1377927 59.16 59.43 2554307 1372626 2554307 1325440 52466149 23.66 1.66 0 3.57 0 0.08 0 0.12 0 0.00 0 7.41 0 2320037 0 100 0 98.85 0 1.42 0 0.01 0 1.21 0 0.01 0 311.71 0 0.26 0 41605 0 2510958 0 89612 0 1912 0 3049 0 0 0 185960 0 116 0 0 0 1635 0 188581 0 1896 0 192228 0 88.83 0 2230425 0 30981 197804 6.384687389045 2510958.0 2320037.0 41605.0 89612.0 1912.0 3049.0 0.0 185960.0 2230425.0 92.4 1.7 3.6 0.1 0.1 0.0 7.4 88.8 50 50 50.00 38 125547900 27.8 21.3 21.5 29.4 0.0 37.3 23.8 smartseq 1445906 SRR4252080 SRP090061 SRS1699754 SRX2172081 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318299: SK_3_B07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318299 GSM2318299 SK_3_B07_smart-seq 183703100 1837031 2016-09-30 15:56:31 126924255 183703100 1837031 2 1837031 index:0,count:1837031,average:50,stdev:0|index:1,count:1837031,average:50,stdev:0 GSM2318299_r1 3.8 2.58 0.11 156788039 199203975 147580790 189173528 127.05 128.18 1683544 1387670 235.096 2110.871 80 6061 69.55 74.05 1951582 1170949 1951582 1170949 70.31 70.79 1951582 1183681 1951582 1119355 22287145 14.21 2.00 0 5.56 0 0.12 0 0.12 0 0.00 0 8.12 0 1683544 0 100 0 98.54 0 1.37 0 0.01 0 1.22 0 0.01 0 220.44 0 0.25 0 36677 0 1837031 0 102227 0 2118 0 2203 0 0 0 149166 0 119 0 0 0 1319 0 177037 0 1258 0 179733 0 86.08 0 1581317 0 37057 181817 4.906414442615 1837031.0 1683544.0 36677.0 102227.0 2118.0 2203.0 0.0 149166.0 1581317.0 91.6 2.0 5.6 0.1 0.1 0.0 8.1 86.1 50 50 50.00 38 91851550 26.8 22.4 22.4 28.4 0.0 37.4 24.2 smartseq 1445937 SRR4251081 SRP090061 SRS1698758 SRX2171082 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317300: 26Dp4_G04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317300 GSM2317300 26Dp4_G04_smart-seq 194379500 1943795 2016-09-30 15:56:31 138541079 194379500 1943795 2 1943795 index:0,count:1943795,average:50,stdev:0|index:1,count:1943795,average:50,stdev:0 GSM2317300_r1 1.14 3.16 0.15 171211714 212355039 162821363 203671191 124.03 125.09 1794971 1474686 265.409 2616.657 113 5339 67.32 70.87 2050818 1208294 2050818 1208294 67.01 67.68 2050818 1202818 2050818 1153801 29975298 17.51 1.71 0 4.63 0 0.08 0 0.11 0 0.00 0 7.46 0 1794971 0 100 0 98.76 0 1.41 0 0.01 0 1.22 0 0.01 0 318.08 0 0.28 0 33244 0 1943795 0 90087 0 1537 0 2235 0 0 0 145052 0 117 0 0 0 1505 0 178647 0 1461 0 181730 0 87.71 0 1704884 0 33298 186049 5.587392636194 1943795.0 1794971.0 33244.0 90087.0 1537.0 2235.0 0.0 145052.0 1704884.0 92.3 1.7 4.6 0.1 0.1 0.0 7.5 87.7 50 50 50.00 38 97189750 27.2 22.0 22.2 28.6 0.0 36.9 23.0 smartseq 1445938 SRR4252081 SRP090061 SRS1699759 SRX2172082 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318300: SK_3_B08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318300 GSM2318300 SK_3_B08_smart-seq 118901200 1189012 2016-09-30 15:56:31 83288391 118901200 1189012 2 1189012 index:0,count:1189012,average:50,stdev:0|index:1,count:1189012,average:50,stdev:0 GSM2318300_r1 7.14 3.07 0.08 103187918 139496474 95839802 131326449 135.19 137.03 1099725 863195 256.088 2708.864 90 3491 78.25 84.48 1323963 860582 1323963 860582 79.68 80.91 1323963 876261 1323963 824167 8859449 8.59 2.18 0 6.82 0 0.07 0 0.07 0 0.00 0 7.37 0 1099725 0 100 0 98.46 0 1.33 0 0.01 0 1.21 0 0.01 0 203.83 0 0.27 0 25946 0 1189012 0 81059 0 860 0 785 0 0 0 87642 0 102 0 0 0 942 0 135586 0 810 0 137440 0 85.67 0 1018666 0 38327 138113 3.603543194093 1189012.0 1099725.0 25946.0 81059.0 860.0 785.0 0.0 87642.0 1018666.0 92.5 2.2 6.8 0.1 0.1 0.0 7.4 85.7 50 50 50.00 38 59450600 26.5 22.8 22.9 27.8 0.0 37.2 24.2 smartseq 1445970 SRR4251082 SRP090061 SRS1698759 SRX2171083 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317301: 26Dp4_G05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317301 GSM2317301 26Dp4_G05_smart-seq 225482500 2254825 2016-09-30 15:56:31 157132131 225482500 2254825 2 2254825 index:0,count:2254825,average:50,stdev:0|index:1,count:2254825,average:50,stdev:0 GSM2317301_r1 2.61 2.86 0.09 200326535 251605050 190890441 241287212 125.6 126.4 2096540 1716131 270.715 2556.674 110 6136 67.89 71.34 2364814 1423359 2364814 1423359 67.52 67.98 2364814 1415620 2364814 1356383 34462830 17.20 1.80 0 4.50 0 0.09 0 0.12 0 0.00 0 6.81 0 2096540 0 100 0 98.74 0 1.42 0 0.01 0 1.21 0 0.01 0 338.22 0 0.26 0 40665 0 2254825 0 101379 0 2050 0 2678 0 0 0 153557 0 159 0 0 0 1764 0 210482 0 1671 0 214076 0 88.48 0 1995161 0 37645 218569 5.806056581219 2254825.0 2096540.0 40665.0 101379.0 2050.0 2678.0 0.0 153557.0 1995161.0 93.0 1.8 4.5 0.1 0.1 0.0 6.8 88.5 50 50 50.00 38 112741250 27.2 22.0 22.2 28.6 0.0 37.2 23.6 smartseq 1445971 SRR4252082 SRP090061 SRS1699757 SRX2172083 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318301: SK_3_B09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318301 GSM2318301 SK_3_B09_smart-seq 124614500 1246145 2016-09-30 15:56:31 85086154 124614500 1246145 2 1246145 index:0,count:1246145,average:50,stdev:0|index:1,count:1246145,average:50,stdev:0 GSM2318301_r1 0.9 0.42 0.12 83498896 75680991 67124444 68126938 90.64 101.49 952911 922282 154.539 932.492 57 6743 65.36 81.26 2120306 622819 2120306 622819 67.31 80.54 2120306 641425 2120306 617304 5699560 6.83 1.80 0 14.96 0 0.32 0 0.32 0 0.00 0 22.89 0 952911 0 100 0 97.53 0 1.47 0 0.01 0 1.15 0 0.01 0 236.11 0 0.22 0 22417 0 1246145 0 186484 0 4042 0 4007 0 0 0 285185 0 9 0 0 0 215 0 23297 0 290 0 23811 0 61.50 0 766427 0 2584 23477 9.085526315789 1246145.0 952911.0 22417.0 186484.0 4042.0 4007.0 0.0 285185.0 766427.0 76.5 1.8 15.0 0.3 0.3 0.0 22.9 61.5 50 50 50.00 38 62307250 25.1 22.5 22.3 30.1 0.0 36.9 19.9 smartseq 1446003 SRR4251083 SRP090061 SRS1698760 SRX2171084 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317302: 26Dp4_G06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317302 GSM2317302 26Dp4_G06_smart-seq 213391100 2133911 2016-09-30 15:56:31 148708505 213391100 2133911 2 2133911 index:0,count:2133911,average:50,stdev:0|index:1,count:2133911,average:50,stdev:0 GSM2317302_r1 4.08 3.27 0.13 185839403 233868363 176792441 224356834 125.84 126.9 1951960 1620983 265.203 2554.349 110 6078 67.55 71.11 2222371 1318487 2222371 1318487 67.54 68.21 2222371 1318302 2222371 1264743 32333990 17.40 1.78 0 4.59 0 0.11 0 0.14 0 0.00 0 8.28 0 1951960 0 100 0 98.71 0 1.36 0 0.01 0 1.20 0 0.01 0 284.52 0 0.27 0 38062 0 2133911 0 97840 0 2290 0 3058 0 0 0 176603 0 105 0 0 0 1582 0 181572 0 1555 0 184814 0 86.89 0 1854120 0 34963 189034 5.406687069187 2133911.0 1951960.0 38062.0 97840.0 2290.0 3058.0 0.0 176603.0 1854120.0 91.5 1.8 4.6 0.1 0.1 0.0 8.3 86.9 50 50 50.00 38 106695550 27.4 21.8 21.9 28.9 0.0 37.2 23.4 smartseq 1446004 SRR4252083 SRP090061 SRS1699762 SRX2172084 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318302: SK_3_B10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318302 GSM2318302 SK_3_B10_smart-seq 219719000 2197190 2016-09-30 15:56:31 150344755 219719000 2197190 2 2197190 index:0,count:2197190,average:50,stdev:0|index:1,count:2197190,average:50,stdev:0 GSM2318302_r1 6.72 2.91 0.14 189717525 248027410 177946714 234571724 130.74 131.82 2045032 1741466 209.860 1883.750 81 8719 73.79 78.82 2382761 1508958 2382761 1508958 74.6 75.42 2382761 1525492 2382761 1443771 22958848 12.10 1.89 0 5.95 0 0.08 0 0.11 0 0.00 0 6.74 0 2045032 0 100 0 98.56 0 1.31 0 0.01 0 1.20 0 0.01 0 282.50 0 0.24 0 41538 0 2197190 0 130665 0 1755 0 2314 0 0 0 148089 0 202 0 0 0 1603 0 204013 0 1517 0 207335 0 87.13 0 1914367 0 34007 212381 6.245214220602 2197190.0 2045032.0 41538.0 130665.0 1755.0 2314.0 0.0 148089.0 1914367.0 93.1 1.9 5.9 0.1 0.1 0.0 6.7 87.1 50 50 50.00 38 109859500 26.7 22.6 22.7 28.0 0.0 37.5 24.7 smartseq 1446033 SRR4251084 SRP090061 SRS1698762 SRX2171085 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317303: 26Dp4_G07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317303 GSM2317303 26Dp4_G07_smart-seq 264101700 2641017 2016-09-30 15:56:31 183031063 264101700 2641017 2 2641017 index:0,count:2641017,average:50,stdev:0|index:1,count:2641017,average:50,stdev:0 GSM2317303_r1 1.39 3.27 0.13 229171440 278736031 219384947 268331534 121.63 122.31 2409514 2016466 255.097 2328.927 105 7643 64.61 67.57 2691926 1556734 2691926 1556734 64.28 64.63 2691926 1548801 2691926 1488911 44372572 19.36 1.89 0 4.00 0 0.08 0 0.13 0 0.00 0 8.56 0 2409514 0 100 0 98.74 0 1.42 0 0.01 0 1.23 0 0.01 0 327.85 0 0.25 0 49890 0 2641017 0 105737 0 2104 0 3384 0 0 0 226015 0 231 0 0 0 1789 0 227111 0 1898 0 231029 0 87.23 0 2303777 0 32327 236491 7.315587589322 2641017.0 2409514.0 49890.0 105737.0 2104.0 3384.0 0.0 226015.0 2303777.0 91.2 1.9 4.0 0.1 0.1 0.0 8.6 87.2 50 50 50.00 38 132050850 27.4 21.7 21.9 28.9 0.0 37.2 23.7 smartseq 1446034 SRR4252084 SRP090061 SRS1699761 SRX2172085 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318303: SK_3_B11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318303 GSM2318303 SK_3_B11_smart-seq 238317600 2383176 2016-09-30 15:56:31 163048191 238317600 2383176 2 2383176 index:0,count:2383176,average:50,stdev:0|index:1,count:2383176,average:50,stdev:0 GSM2318303_r1 9.08 2.8 0.1 205569547 276977554 190012213 259288691 134.74 136.46 2212519 1823347 218.462 1955.159 81 8941 77.82 84.41 2668722 1721837 2668722 1721837 79.72 80.81 2668722 1763891 2668722 1648409 17222171 8.38 2.08 0 7.24 0 0.09 0 0.09 0 0.00 0 6.99 0 2212519 0 100 0 98.48 0 1.29 0 0.01 0 1.17 0 0.01 0 408.54 0 0.25 0 49466 0 2383176 0 172615 0 2044 0 2131 0 0 0 166482 0 222 0 0 0 1720 0 258251 0 1403 0 261596 0 85.60 0 2039904 0 37507 265640 7.082411283227 2383176.0 2212519.0 49466.0 172615.0 2044.0 2131.0 0.0 166482.0 2039904.0 92.8 2.1 7.2 0.1 0.1 0.0 7.0 85.6 50 50 50.00 38 119158800 26.5 22.8 23.0 27.6 0.0 37.5 24.8 smartseq 1446065 SRR4251085 SRP090061 SRS1698761 SRX2171086 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317304: 26Dp4_G08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317304 GSM2317304 26Dp4_G08_smart-seq 132691000 1326910 2016-09-30 15:56:31 92644110 132691000 1326910 2 1326910 index:0,count:1326910,average:50,stdev:0|index:1,count:1326910,average:50,stdev:0 GSM2317304_r1 2.45 3.64 0.22 116594982 143824898 111252083 138221234 123.35 124.24 1221855 1005747 275.546 2809.273 110 3480 65.27 68.49 1376506 797467 1376506 797467 65.37 65.91 1376506 798692 1376506 767371 22606634 19.39 1.84 0 4.34 0 0.09 0 0.12 0 0.00 0 7.71 0 1221855 0 100 0 98.62 0 1.37 0 0.01 0 1.22 0 0.01 0 265.38 0 0.27 0 24430 0 1326910 0 57558 0 1161 0 1546 0 0 0 102348 0 81 0 0 0 1121 0 111623 0 983 0 113808 0 87.74 0 1164297 0 30291 116281 3.838797002410 1326910.0 1221855.0 24430.0 57558.0 1161.0 1546.0 0.0 102348.0 1164297.0 92.1 1.8 4.3 0.1 0.1 0.0 7.7 87.7 50 50 50.00 38 66345500 27.7 21.5 21.8 29.0 0.0 37.2 24.0 smartseq 1446066 SRR4252085 SRP090061 SRS1699758 SRX2172086 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318304: SK_3_B12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318304 GSM2318304 SK_3_B12_smart-seq 244023900 2440239 2016-09-30 15:56:31 166057974 244023900 2440239 2 2440239 index:0,count:2440239,average:50,stdev:0|index:1,count:2440239,average:50,stdev:0 GSM2318304_r1 3.78 3.25 0.18 201753021 252005844 190283515 239956677 124.91 126.1 2203460 1925448 191.631 1750.085 81 10616 70.11 74.47 2552055 1544789 2552055 1544789 70.34 71.22 2552055 1549977 2552055 1477339 29355639 14.55 1.90 0 5.29 0 0.13 0 0.09 0 0.00 0 9.49 0 2203460 0 100 0 98.48 0 1.32 0 0.01 0 1.18 0 0.01 0 302.93 0 0.23 0 46436 0 2440239 0 129113 0 3072 0 2216 0 0 0 231491 0 99 0 0 0 1335 0 203206 0 1698 0 206338 0 85.01 0 2074347 0 27837 211124 7.584294284585 2440239.0 2203460.0 46436.0 129113.0 3072.0 2216.0 0.0 231491.0 2074347.0 90.3 1.9 5.3 0.1 0.1 0.0 9.5 85.0 50 50 50.00 38 122011950 26.9 22.2 22.3 28.5 0.0 37.5 24.0 smartseq 1446097 SRR4251086 SRP090061 SRS1698764 SRX2171087 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317305: 26Dp4_G09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317305 GSM2317305 26Dp4_G09_smart-seq 76050400 760504 2016-09-30 15:56:31 52942516 76050400 760504 2 760504 index:0,count:760504,average:50,stdev:0|index:1,count:760504,average:50,stdev:0 GSM2317305_r1 1.79 3.47 0.16 67228202 82340962 64169388 79168469 122.48 123.37 701421 575998 293.817 2555.592 100 1810 61.72 64.76 791205 432887 791205 432887 61.73 62.11 791205 432981 791205 415212 14698161 21.86 1.91 0 4.33 0 0.08 0 0.13 0 0.00 0 7.56 0 701421 0 100 0 98.68 0 1.41 0 0.01 0 1.22 0 0.01 0 210.60 0 0.27 0 14505 0 760504 0 32925 0 588 0 1005 0 0 0 57490 0 68 0 0 0 517 0 62442 0 595 0 63622 0 87.90 0 668496 0 27267 63649 2.334286866909 760504.0 701421.0 14505.0 32925.0 588.0 1005.0 0.0 57490.0 668496.0 92.2 1.9 4.3 0.1 0.1 0.0 7.6 87.9 50 50 50.00 38 38025200 27.8 21.4 21.6 29.2 0.0 37.2 23.9 smartseq 1446098 SRR4252086 SRP090061 SRS1699760 SRX2172087 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318305: SK_3_C01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318305 GSM2318305 SK_3_C01_smart-seq 177176200 1771762 2016-09-30 15:56:31 121458662 177176200 1771762 2 1771762 index:0,count:1771762,average:50,stdev:0|index:1,count:1771762,average:50,stdev:0 GSM2318305_r1 12.19 2.03 0.07 136017211 185802354 114615500 163234062 136.6 142.42 1503246 1336128 196.324 1001.327 81 7481 83.04 99.04 2274405 1248281 2274405 1248281 90.27 95.31 2274405 1357053 2274405 1201251 379006 0.28 2.95 0 13.71 0 0.10 0 0.00 0 0.00 0 15.05 0 1503246 0 100 0 97.86 0 1.18 0 0.01 0 1.12 0 0.00 0 318.92 0 0.26 0 52270 0 1771762 0 242897 0 1760 0 83 0 0 0 266673 0 93 0 0 0 1038 0 138738 0 663 0 140532 0 71.14 0 1260349 0 8972 142522 15.885198395007 1771762.0 1503246.0 52270.0 242897.0 1760.0 83.0 0.0 266673.0 1260349.0 84.8 3.0 13.7 0.1 0.0 0.0 15.1 71.1 50 50 50.00 38 88588100 25.3 23.2 23.2 28.3 0.0 37.2 22.0 smartseq 1446128 SRR4251087 SRP090061 SRS1698763 SRX2171088 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317306: 26Dp4_G10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317306 GSM2317306 26Dp4_G10_smart-seq 130127600 1301276 2016-09-30 15:56:31 90975003 130127600 1301276 2 1301276 index:0,count:1301276,average:50,stdev:0|index:1,count:1301276,average:50,stdev:0 GSM2317306_r1 3.67 3.09 0.2 113394630 139130003 108099198 133873472 122.7 123.84 1188288 1010701 278.454 2420.439 110 3476 60.64 63.71 1357942 720603 1357942 720603 60.69 61.27 1357942 721191 1357942 692977 25212605 22.23 1.93 0 4.40 0 0.10 0 0.15 0 0.00 0 8.43 0 1188288 0 100 0 98.68 0 1.40 0 0.01 0 1.22 0 0.01 0 260.26 0 0.28 0 25066 0 1301276 0 57272 0 1340 0 1959 0 0 0 109689 0 77 0 0 0 818 0 87221 0 1037 0 89153 0 86.92 0 1131016 0 26593 90274 3.394652728162 1301276.0 1188288.0 25066.0 57272.0 1340.0 1959.0 0.0 109689.0 1131016.0 91.3 1.9 4.4 0.1 0.2 0.0 8.4 86.9 50 50 50.00 38 65063800 27.9 21.3 21.5 29.3 0.0 37.2 23.8 smartseq 1446129 SRR4252087 SRP090061 SRS1699763 SRX2172088 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318306: SK_3_C02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318306 GSM2318306 SK_3_C02_smart-seq 198832700 1988327 2016-09-30 15:56:31 135525660 198832700 1988327 2 1988327 index:0,count:1988327,average:50,stdev:0|index:1,count:1988327,average:50,stdev:0 GSM2318306_r1 1.4 3.11 0.26 165863644 205689273 156132834 195705274 124.01 125.35 1808337 1568102 202.432 1663.231 70 8069 67.39 71.71 2115290 1218589 2115290 1218589 68.08 68.74 2115290 1231129 2115290 1168026 25672213 15.48 2.02 0 5.49 0 0.15 0 0.13 0 0.00 0 8.78 0 1808337 0 100 0 98.45 0 1.39 0 0.01 0 1.18 0 0.01 0 298.25 0 0.23 0 40185 0 1988327 0 109103 0 3000 0 2486 0 0 0 174504 0 90 0 0 0 1269 0 164480 0 1355 0 167194 0 85.46 0 1699234 0 27177 169537 6.238252934467 1988327.0 1808337.0 40185.0 109103.0 3000.0 2486.0 0.0 174504.0 1699234.0 90.9 2.0 5.5 0.2 0.1 0.0 8.8 85.5 50 50 50.00 38 99416350 26.9 22.1 22.2 28.7 0.0 37.5 24.1 smartseq 1446160 SRR4251088 SRP090061 SRS1698766 SRX2171089 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317307: 26Dp4_G11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317307 GSM2317307 26Dp4_G11_smart-seq 220190300 2201903 2016-09-30 15:56:31 152279541 220190300 2201903 2 2201903 index:0,count:2201903,average:50,stdev:0|index:1,count:2201903,average:50,stdev:0 GSM2317307_r1 1.81 3.0 0.16 188783403 233026825 180526585 224054710 123.44 124.11 2006396 1716637 245.334 2250.932 100 6837 64.87 67.93 2243348 1301517 2243348 1301517 64.78 65.16 2243348 1299654 2243348 1248452 36721423 19.45 1.82 0 4.11 0 0.08 0 0.15 0 0.00 0 8.66 0 2006396 0 100 0 98.69 0 1.40 0 0.01 0 1.21 0 0.01 0 396.34 0 0.29 0 40084 0 2201903 0 90422 0 1723 0 3206 0 0 0 190578 0 97 0 0 0 1510 0 167876 0 1738 0 171221 0 87.01 0 1915974 0 28028 174401 6.222384758099 2201903.0 2006396.0 40084.0 90422.0 1723.0 3206.0 0.0 190578.0 1915974.0 91.1 1.8 4.1 0.1 0.1 0.0 8.7 87.0 50 50 50.00 38 110095150 27.4 21.6 21.7 29.2 0.0 37.3 23.6 smartseq 1446161 SRR4252088 SRP090061 SRS1699765 SRX2172089 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318307: SK_3_C03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318307 GSM2318307 SK_3_C03_smart-seq 128482700 1284827 2016-09-30 15:56:31 88343570 128482700 1284827 2 1284827 index:0,count:1284827,average:50,stdev:0|index:1,count:1284827,average:50,stdev:0 GSM2318307_r1 5.57 1.41 0.08 92295794 113845581 77713084 100341564 123.35 129.12 1032034 954589 182.851 817.765 57 5924 82.79 98.71 1681412 854472 1681412 854472 88.75 95.44 1681412 915972 1681412 826096 357353 0.39 2.70 0 12.95 0 0.11 0 0.00 0 0.00 0 19.57 0 1032034 0 100 0 97.79 0 1.24 0 0.01 0 1.12 0 0.00 0 272.08 0 0.24 0 34657 0 1284827 0 166423 0 1359 0 50 0 0 0 251384 0 48 0 0 0 427 0 67736 0 277 0 68488 0 67.37 0 865611 0 3812 69196 18.152151101784 1284827.0 1032034.0 34657.0 166423.0 1359.0 50.0 0.0 251384.0 865611.0 80.3 2.7 13.0 0.1 0.0 0.0 19.6 67.4 50 50 50.00 38 64241350 24.9 23.0 22.7 29.3 0.0 37.0 20.5 smartseq 1446192 SRR4251089 SRP090061 SRS1698765 SRX2171090 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317308: 26Dp4_H01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317308 GSM2317308 26Dp4_H01_smart-seq 110042700 1100427 2016-09-30 15:56:31 78206339 110042700 1100427 2 1100427 index:0,count:1100427,average:50,stdev:0|index:1,count:1100427,average:50,stdev:0 GSM2317308_r1 5.05 3.2 0.15 98863129 125757262 93600894 120530856 127.2 128.77 1022931 820148 311.309 2957.383 145 2332 68.07 71.99 1185971 696316 1185971 696316 68.21 69.01 1185971 697785 1185971 667506 15659152 15.84 1.86 0 5.06 0 0.09 0 0.10 0 0.00 0 6.85 0 1022931 0 100 0 98.80 0 1.38 0 0.01 0 1.23 0 0.01 0 208.50 0 0.30 0 20418 0 1100427 0 55731 0 977 0 1140 0 0 0 75379 0 88 0 0 0 746 0 99169 0 812 0 100815 0 87.89 0 967200 0 34498 102495 2.971041799525 1100427.0 1022931.0 20418.0 55731.0 977.0 1140.0 0.0 75379.0 967200.0 93.0 1.9 5.1 0.1 0.1 0.0 6.8 87.9 50 50 50.00 38 55021350 27.1 22.2 22.4 28.3 0.0 36.9 23.2 smartseq 1446193 SRR4252089 SRP090061 SRS1699764 SRX2172090 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318308: SK_3_C04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318308 GSM2318308 SK_3_C04_smart-seq 126858500 1268585 2016-09-30 15:56:31 87479611 126858500 1268585 2 1268585 index:0,count:1268585,average:50,stdev:0|index:1,count:1268585,average:50,stdev:0 GSM2318308_r1 1.84 2.91 0.07 107416741 136606047 101699715 130162305 127.17 127.99 1149830 924975 243.012 2249.313 88 3917 74.78 79.14 1305481 859857 1305481 859857 75.32 75.96 1305481 866032 1305481 825321 12918625 12.03 1.99 0 4.99 0 0.11 0 0.08 0 0.00 0 9.17 0 1149830 0 100 0 98.52 0 1.37 0 0.01 0 1.21 0 0.01 0 240.36 0 0.24 0 25214 0 1268585 0 63317 0 1376 0 1039 0 0 0 116340 0 68 0 0 0 953 0 133456 0 954 0 135431 0 85.65 0 1086513 0 32055 136473 4.257463734207 1268585.0 1149830.0 25214.0 63317.0 1376.0 1039.0 0.0 116340.0 1086513.0 90.6 2.0 5.0 0.1 0.1 0.0 9.2 85.6 50 50 50.00 38 63429250 26.9 22.3 22.4 28.4 0.0 37.4 24.2 smartseq 1446416 SRR4251090 SRP090061 SRS1698767 SRX2171091 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317309: 26Dp4_H02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317309 GSM2317309 26Dp4_H02_smart-seq 67723900 677239 2016-09-30 15:56:31 47662325 67723900 677239 2 677239 index:0,count:677239,average:50,stdev:0|index:1,count:677239,average:50,stdev:0 GSM2317309_r1 3.02 3.5 0.13 60206923 74545835 57556396 71800185 123.82 124.75 625537 512648 290.376 3001.221 148 1604 65.54 68.64 702939 410004 702939 410004 65.52 66.08 702939 409858 702939 394707 11970627 19.88 1.73 0 4.16 0 0.09 0 0.08 0 0.00 0 7.47 0 625537 0 100 0 98.81 0 1.38 0 0.01 0 1.25 0 0.01 0 152.38 0 0.28 0 11706 0 677239 0 28177 0 623 0 523 0 0 0 50556 0 27 0 0 0 530 0 56421 0 445 0 57423 0 88.21 0 597360 0 22082 58296 2.639978262839 677239.0 625537.0 11706.0 28177.0 623.0 523.0 0.0 50556.0 597360.0 92.4 1.7 4.2 0.1 0.1 0.0 7.5 88.2 50 50 50.00 38 33861950 27.6 21.6 21.7 29.1 0.0 37.1 23.3 smartseq 1446417 SRR4252090 SRP090061 SRS1699769 SRX2172091 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318309: SK_3_C05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318309 GSM2318309 SK_3_C05_smart-seq 30076300 300763 2016-09-30 15:56:31 20831835 30076300 300763 2 300763 index:0,count:300763,average:50,stdev:0|index:1,count:300763,average:50,stdev:0 GSM2318309_r1 0.4 6.38 0.71 623860 447653 552445 402704 71.76 72.89 8087 6859 143.877 15097.425 51 343 10.83 12.6 10372 876 10372 876 12.71 12.21 10372 1028 10372 849 246721 39.55 0.32 0 0.38 0 0.03 0 0.04 0 0.00 0 97.25 0 8087 0 100 0 91.22 0 1.09 0 0.01 0 1.08 0 0.00 0 49.22 0 0.56 0 948 0 300763 0 1132 0 76 0 108 0 0 0 292492 0 0 0 0 0 0 0 50 0 19 0 69 0 2.31 0 6955 0 49 49 1.000000000000 300763.0 8087.0 948.0 1132.0 76.0 108.0 0.0 292492.0 6955.0 2.7 0.3 0.4 0.0 0.0 0.0 97.2 2.3 50 50 50.00 38 15038150 21.6 22.1 21.3 35.1 0.0 34.9 15.2 smartseq 1446451 SRR4251091 SRP090061 SRS1698768 SRX2171092 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317310: 26Dp4_H03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317310 GSM2317310 26Dp4_H03_smart-seq 177252000 1772520 2016-09-30 15:56:31 124836831 177252000 1772520 2 1772520 index:0,count:1772520,average:50,stdev:0|index:1,count:1772520,average:50,stdev:0 GSM2317310_r1 3.49 3.29 0.12 157044030 201694011 149251373 193002361 128.43 129.31 1643298 1340503 275.626 2650.811 110 4780 70.92 74.73 1859213 1165478 1859213 1165478 71.08 71.73 1859213 1167994 1859213 1118685 23907088 15.22 1.82 0 4.72 0 0.07 0 0.09 0 0.00 0 7.13 0 1643298 0 100 0 98.72 0 1.33 0 0.01 0 1.22 0 0.01 0 290.05 0 0.28 0 32316 0 1772520 0 83700 0 1270 0 1551 0 0 0 126401 0 127 0 0 0 1338 0 166808 0 1234 0 169507 0 87.99 0 1559598 0 36110 173205 4.796593741346 1772520.0 1643298.0 32316.0 83700.0 1270.0 1551.0 0.0 126401.0 1559598.0 92.7 1.8 4.7 0.1 0.1 0.0 7.1 88.0 50 50 50.00 38 88626000 27.2 22.0 22.1 28.7 0.0 37.1 23.5 smartseq 1446452 SRR4252091 SRP090061 SRS1699771 SRX2172092 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318310: SK_3_C06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318310 GSM2318310 SK_3_C06_smart-seq 135399200 1353992 2016-09-30 15:56:31 93522037 135399200 1353992 2 1353992 index:0,count:1353992,average:50,stdev:0|index:1,count:1353992,average:50,stdev:0 GSM2318310_r1 5.51 2.7 0.1 115327507 148695395 106496113 139549546 128.93 131.04 1235172 1027137 245.070 2071.353 81 4264 69.95 75.97 1529289 864043 1529289 864043 71.62 72.62 1529289 884680 1529289 825949 15002968 13.01 2.13 0 7.23 0 0.12 0 0.09 0 0.00 0 8.56 0 1235172 0 100 0 98.50 0 1.33 0 0.01 0 1.19 0 0.01 0 232.11 0 0.25 0 28839 0 1353992 0 97826 0 1577 0 1274 0 0 0 115969 0 51 0 0 0 911 0 122753 0 979 0 124694 0 84.00 0 1137346 0 29966 126748 4.229727023960 1353992.0 1235172.0 28839.0 97826.0 1577.0 1274.0 0.0 115969.0 1137346.0 91.2 2.1 7.2 0.1 0.1 0.0 8.6 84.0 50 50 50.00 38 67699600 26.7 22.3 22.4 28.6 0.0 37.3 23.6 smartseq 1446481 SRR4251092 SRP090061 SRS1698769 SRX2171093 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317311: 26Dp4_H04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317311 GSM2317311 26Dp4_H04_smart-seq 194739500 1947395 2016-09-30 15:56:31 135552407 194739500 1947395 2 1947395 index:0,count:1947395,average:50,stdev:0|index:1,count:1947395,average:50,stdev:0 GSM2317311_r1 4.55 3.19 0.1 171113917 216064887 162461338 207040599 126.27 127.44 1789859 1503932 263.471 2492.692 137 5384 65.9 69.52 2040918 1179576 2040918 1179576 66.3 66.87 2040918 1186693 2040918 1134688 30605444 17.89 1.75 0 4.78 0 0.06 0 0.10 0 0.00 0 7.93 0 1789859 0 100 0 98.78 0 1.37 0 0.01 0 1.19 0 0.01 0 333.84 0 0.27 0 34003 0 1947395 0 93038 0 1210 0 1914 0 0 0 154412 0 100 0 0 0 1334 0 156453 0 1495 0 159382 0 87.13 0 1696821 0 27161 164553 6.058429365635 1947395.0 1789859.0 34003.0 93038.0 1210.0 1914.0 0.0 154412.0 1696821.0 91.9 1.7 4.8 0.1 0.1 0.0 7.9 87.1 50 50 50.00 38 97369750 27.5 21.7 21.8 29.1 0.0 37.2 23.5 smartseq 1446482 SRR4252092 SRP090061 SRS1699767 SRX2172093 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318311: SK_3_C07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318311 GSM2318311 SK_3_C07_smart-seq 218272200 2182722 2016-09-30 15:56:31 149439001 218272200 2182722 2 2182722 index:0,count:2182722,average:50,stdev:0|index:1,count:2182722,average:50,stdev:0 GSM2318311_r1 2.73 2.63 0.1 185173862 236061586 174210329 223840166 127.48 128.49 1997267 1691359 221.116 2029.318 79 7818 70.18 74.75 2318730 1401722 2318730 1401722 70.49 71.07 2318730 1407926 2318730 1332748 27048026 14.61 1.92 0 5.59 0 0.10 0 0.12 0 0.00 0 8.27 0 1997267 0 100 0 98.54 0 1.39 0 0.01 0 1.20 0 0.01 0 291.03 0 0.24 0 41885 0 2182722 0 122006 0 2140 0 2725 0 0 0 180590 0 158 0 0 0 1669 0 192518 0 1551 0 195896 0 85.91 0 1875261 0 33050 201169 6.086807866868 2182722.0 1997267.0 41885.0 122006.0 2140.0 2725.0 0.0 180590.0 1875261.0 91.5 1.9 5.6 0.1 0.1 0.0 8.3 85.9 50 50 50.00 38 109136100 26.7 22.4 22.4 28.5 0.0 37.4 23.9 smartseq 1446515 SRR4251093 SRP090061 SRS1698770 SRX2171094 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317312: 26Dp4_H05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317312 GSM2317312 26Dp4_H05_smart-seq 243839400 2438394 2016-09-30 15:56:31 169414219 243839400 2438394 2 2438394 index:0,count:2438394,average:50,stdev:0|index:1,count:2438394,average:50,stdev:0 GSM2317312_r1 1.08 3.04 0.2 217342992 264918347 208252577 255049102 121.89 122.47 2261073 1899272 274.909 2460.800 137 6520 63.07 65.88 2491003 1426048 2491003 1426048 62.89 63.17 2491003 1421962 2491003 1367452 44706175 20.57 1.65 0 3.95 0 0.07 0 0.14 0 0.00 0 7.06 0 2261073 0 100 0 98.85 0 1.43 0 0.01 0 1.21 0 0.01 0 337.62 0 0.26 0 40355 0 2438394 0 96404 0 1740 0 3327 0 0 0 172254 0 147 0 0 0 1632 0 193097 0 1813 0 196689 0 88.77 0 2164669 0 30056 203213 6.761145861059 2438394.0 2261073.0 40355.0 96404.0 1740.0 3327.0 0.0 172254.0 2164669.0 92.7 1.7 4.0 0.1 0.1 0.0 7.1 88.8 50 50 50.00 38 121919700 27.7 21.4 21.6 29.3 0.0 37.2 23.5 smartseq 1446516 SRR4252093 SRP090061 SRS1699768 SRX2172094 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318312: SK_3_C08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318312 GSM2318312 SK_3_C08_smart-seq 141668800 1416688 2016-09-30 15:56:31 98070115 141668800 1416688 2 1416688 index:0,count:1416688,average:50,stdev:0|index:1,count:1416688,average:50,stdev:0 GSM2318312_r1 8.45 2.79 0.1 119707677 158746806 111184094 149089377 132.61 134.09 1283468 1064633 239.086 2169.546 81 4519 72.95 78.74 1525210 936275 1525210 936275 74.86 75.57 1525210 960748 1525210 898485 13834139 11.56 2.06 0 6.67 0 0.10 0 0.08 0 0.00 0 9.22 0 1283468 0 100 0 98.50 0 1.32 0 0.01 0 1.21 0 0.01 0 255.00 0 0.26 0 29131 0 1416688 0 94461 0 1416 0 1182 0 0 0 130622 0 114 0 0 0 984 0 130608 0 811 0 132517 0 83.93 0 1189007 0 33084 134519 4.065983557006 1416688.0 1283468.0 29131.0 94461.0 1416.0 1182.0 0.0 130622.0 1189007.0 90.6 2.1 6.7 0.1 0.1 0.0 9.2 83.9 50 50 50.00 38 70834400 26.7 22.5 22.5 28.3 0.0 37.3 24.0 smartseq 1446547 SRR4251094 SRP090061 SRS1698774 SRX2171095 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317313: 26Dp4_H06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317313 GSM2317313 26Dp4_H06_smart-seq 42960500 429605 2016-09-30 15:56:31 29466581 42960500 429605 2 429605 index:0,count:429605,average:50,stdev:0|index:1,count:429605,average:50,stdev:0 GSM2317313_r1 5.21 3.04 0.19 36591234 46547022 34333961 44318539 127.21 129.08 400352 360690 161.804 1389.617 100 2419 68.44 73.1 484559 274018 484559 274018 68.0 69.31 484559 272239 484559 259797 5258311 14.37 1.84 0 5.94 0 0.12 0 0.22 0 0.00 0 6.47 0 400352 0 100 0 98.50 0 1.32 0 0.01 0 1.22 0 0.00 0 140.60 0 0.24 0 7911 0 429605 0 25510 0 511 0 927 0 0 0 27815 0 19 0 0 0 315 0 41088 0 312 0 41734 0 87.25 0 374842 0 17496 41365 2.364254686786 429605.0 400352.0 7911.0 25510.0 511.0 927.0 0.0 27815.0 374842.0 93.2 1.8 5.9 0.1 0.2 0.0 6.5 87.3 50 50 50.00 38 21480250 25.6 23.9 24.0 26.4 0.0 37.5 25.5 smartseq 1446548 SRR4252094 SRP090061 SRS1699772 SRX2172095 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318313: SK_3_C09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318313 GSM2318313 SK_3_C09_smart-seq 155972100 1559721 2016-09-30 15:56:31 106352453 155972100 1559721 2 1559721 index:0,count:1559721,average:50,stdev:0|index:1,count:1559721,average:50,stdev:0 GSM2318313_r1 6.75 2.59 0.08 133695604 172707783 123870929 162430148 129.18 131.13 1437286 1196468 228.217 2023.350 81 5406 71.75 77.65 1773687 1031296 1773687 1031296 73.23 74.58 1773687 1052503 1773687 990492 14804968 11.07 2.00 0 7.00 0 0.12 0 0.12 0 0.00 0 7.61 0 1437286 0 100 0 98.53 0 1.35 0 0.01 0 1.22 0 0.01 0 207.96 0 0.24 0 31209 0 1559721 0 109125 0 1941 0 1825 0 0 0 118669 0 100 0 0 0 1043 0 144541 0 1063 0 146747 0 85.15 0 1328161 0 31456 148373 4.716842573754 1559721.0 1437286.0 31209.0 109125.0 1941.0 1825.0 0.0 118669.0 1328161.0 92.2 2.0 7.0 0.1 0.1 0.0 7.6 85.2 50 50 50.00 38 77986050 26.7 22.5 22.6 28.2 0.0 37.5 24.4 smartseq 1446578 SRR4251095 SRP090061 SRS1698772 SRX2171096 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317314: 26Dp4_H07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317314 GSM2317314 26Dp4_H07_smart-seq 196993600 1969936 2016-09-30 15:56:31 137698222 196993600 1969936 2 1969936 index:0,count:1969936,average:50,stdev:0|index:1,count:1969936,average:50,stdev:0 GSM2317314_r1 1.99 3.0 0.17 174359281 217624501 165853173 208413963 124.81 125.66 1826210 1499801 270.597 2677.988 110 5429 67.33 70.87 2068613 1229556 2068613 1229556 67.17 67.66 2068613 1226718 2068613 1173825 31318086 17.96 1.77 0 4.64 0 0.07 0 0.11 0 0.00 0 7.12 0 1826210 0 100 0 98.75 0 1.39 0 0.01 0 1.22 0 0.01 0 337.70 0 0.27 0 34841 0 1969936 0 91323 0 1418 0 2101 0 0 0 140207 0 159 0 0 0 1524 0 178740 0 1493 0 181916 0 88.07 0 1734887 0 35364 186535 5.274714398824 1969936.0 1826210.0 34841.0 91323.0 1418.0 2101.0 0.0 140207.0 1734887.0 92.7 1.8 4.6 0.1 0.1 0.0 7.1 88.1 50 50 50.00 38 98496800 27.3 21.9 22.0 28.8 0.0 37.1 23.5 smartseq 1446579 SRR4252095 SRP090061 SRS1699770 SRX2172096 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318314: SK_3_C10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318314 GSM2318314 SK_3_C10_smart-seq 95406300 954063 2016-09-30 15:56:31 66431182 95406300 954063 2 954063 index:0,count:954063,average:50,stdev:0|index:1,count:954063,average:50,stdev:0 GSM2318314_r1 15.99 2.15 0.05 82989312 115870622 76840220 109943406 139.62 143.08 885647 748701 252.346 2341.539 83 2960 74.82 81.1 1143465 662612 1143465 662612 75.49 77.85 1143465 668557 1143465 636001 8656921 10.43 1.94 0 7.20 0 0.11 0 0.07 0 0.00 0 6.99 0 885647 0 100 0 98.44 0 1.30 0 0.01 0 1.20 0 0.01 0 190.81 0 0.29 0 18504 0 954063 0 68660 0 1083 0 632 0 0 0 66701 0 40 0 0 0 639 0 73271 0 581 0 74531 0 85.63 0 816987 0 30286 73564 2.428977085122 954063.0 885647.0 18504.0 68660.0 1083.0 632.0 0.0 66701.0 816987.0 92.8 1.9 7.2 0.1 0.1 0.0 7.0 85.6 50 50 50.00 38 47703150 26.7 22.5 22.6 28.2 0.0 37.2 23.9 smartseq 1446610 SRR4251096 SRP090061 SRS1698771 SRX2171097 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317315: 26Dp4_H08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317315 GSM2317315 26Dp4_H08_smart-seq 137545900 1375459 2016-09-30 15:56:31 96819991 137545900 1375459 2 1375459 index:0,count:1375459,average:50,stdev:0|index:1,count:1375459,average:50,stdev:0 GSM2317315_r1 3.57 3.46 0.17 120817710 150901756 114029361 143872752 124.9 126.17 1258448 1017993 286.805 2665.393 134 3326 66.53 70.6 1457244 837308 1457244 837308 67.22 67.8 1457244 845880 1457244 804076 21055598 17.43 1.99 0 5.27 0 0.12 0 0.10 0 0.00 0 8.28 0 1258448 0 100 0 98.70 0 1.37 0 0.01 0 1.21 0 0.01 0 215.29 0 0.29 0 27407 0 1375459 0 72423 0 1667 0 1437 0 0 0 113907 0 106 0 0 0 1032 0 124847 0 1071 0 127056 0 86.23 0 1186025 0 36357 129526 3.562615177270 1375459.0 1258448.0 27407.0 72423.0 1667.0 1437.0 0.0 113907.0 1186025.0 91.5 2.0 5.3 0.1 0.1 0.0 8.3 86.2 50 50 50.00 38 68772950 27.2 21.9 22.1 28.7 0.0 37.1 23.3 smartseq 1446611 SRR4252096 SRP090061 SRS1699773 SRX2172097 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318315: SK_3_C11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318315 GSM2318315 SK_3_C11_smart-seq 237245800 2372458 2016-09-30 15:56:31 161953790 237245800 2372458 2 2372458 index:0,count:2372458,average:50,stdev:0|index:1,count:2372458,average:50,stdev:0 GSM2318315_r1 4.11 3.09 0.1 201703630 260088331 188047774 246218962 128.95 130.93 2185053 1808627 206.823 2143.460 81 9490 74.33 79.9 2656142 1624207 2656142 1624207 74.82 76.23 2656142 1634766 2656142 1549724 22147042 10.98 1.92 0 6.42 0 0.09 0 0.10 0 0.00 0 7.70 0 2185053 0 100 0 98.47 0 1.33 0 0.01 0 1.18 0 0.01 0 406.71 0 0.24 0 45434 0 2372458 0 152227 0 2191 0 2468 0 0 0 182746 0 207 0 0 0 2023 0 259386 0 1520 0 263136 0 85.68 0 2032826 0 39456 266816 6.762368207624 2372458.0 2185053.0 45434.0 152227.0 2191.0 2468.0 0.0 182746.0 2032826.0 92.1 1.9 6.4 0.1 0.1 0.0 7.7 85.7 50 50 50.00 38 118622900 26.5 22.8 23.0 27.7 0.0 37.5 24.7 smartseq 1446641 SRR4251097 SRP090061 SRS1698773 SRX2171098 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317316: 26Dp4_H09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317316 GSM2317316 26Dp4_H09_smart-seq 134915400 1349154 2016-09-30 15:56:31 93948049 134915400 1349154 2 1349154 index:0,count:1349154,average:50,stdev:0|index:1,count:1349154,average:50,stdev:0 GSM2317316_r1 1.46 3.39 0.13 118206182 145838393 112018517 139227122 123.38 124.29 1238650 1016721 277.108 2571.023 110 3546 65.8 69.54 1420557 815072 1420557 815072 66.22 66.7 1420557 820181 1420557 781745 22228732 18.81 1.95 0 4.93 0 0.08 0 0.11 0 0.00 0 8.00 0 1238650 0 100 0 98.56 0 1.40 0 0.01 0 1.23 0 0.01 0 285.70 0 0.26 0 26329 0 1349154 0 66560 0 1081 0 1456 0 0 0 107967 0 111 0 0 0 942 0 115350 0 1049 0 117452 0 86.88 0 1172090 0 28027 119962 4.280229778428 1349154.0 1238650.0 26329.0 66560.0 1081.0 1456.0 0.0 107967.0 1172090.0 91.8 2.0 4.9 0.1 0.1 0.0 8.0 86.9 50 50 50.00 38 67457700 27.4 21.7 21.9 28.9 0.0 37.2 23.6 smartseq 1446642 SRR4252097 SRP090061 SRS1699774 SRX2172098 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318316: SK_3_C12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318316 GSM2318316 SK_3_C12_smart-seq 215928100 2159281 2016-09-30 15:56:31 148197084 215928100 2159281 2 2159281 index:0,count:2159281,average:50,stdev:0|index:1,count:2159281,average:50,stdev:0 GSM2318316_r1 2.8 2.66 0.1 183643525 234509550 172402132 221942662 127.7 128.74 1985385 1685727 211.509 1965.589 81 8385 70.68 75.42 2315775 1403212 2315775 1403212 71.02 71.66 2315775 1410050 2315775 1333263 25859291 14.08 1.88 0 5.78 0 0.10 0 0.13 0 0.00 0 7.82 0 1985385 0 100 0 98.54 0 1.38 0 0.01 0 1.19 0 0.01 0 250.76 0 0.24 0 40592 0 2159281 0 124869 0 2134 0 2823 0 0 0 168939 0 161 0 0 0 1686 0 196903 0 1474 0 200224 0 86.16 0 1860516 0 33014 205662 6.229538983462 2159281.0 1985385.0 40592.0 124869.0 2134.0 2823.0 0.0 168939.0 1860516.0 91.9 1.9 5.8 0.1 0.1 0.0 7.8 86.2 50 50 50.00 38 107964050 26.7 22.5 22.6 28.2 0.0 37.4 24.2 smartseq 1446673 SRR4251098 SRP090061 SRS1698775 SRX2171099 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317317: 26Dp4_H10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317317 GSM2317317 26Dp4_H10_smart-seq 136512400 1365124 2016-09-30 15:56:31 95883554 136512400 1365124 2 1365124 index:0,count:1365124,average:50,stdev:0|index:1,count:1365124,average:50,stdev:0 GSM2317317_r1 0.51 3.54 0.22 120446974 148425135 115471325 143092075 123.23 123.92 1258600 1024961 277.224 2813.237 110 3638 67.66 70.65 1387473 851622 1387473 851622 67.76 68.2 1387473 852818 1387473 822132 21845524 18.14 1.77 0 3.89 0 0.08 0 0.13 0 0.00 0 7.60 0 1258600 0 100 0 98.68 0 1.42 0 0.01 0 1.22 0 0.01 0 258.66 0 0.28 0 24143 0 1365124 0 53164 0 1043 0 1788 0 0 0 103693 0 100 0 0 0 1004 0 118340 0 1054 0 120498 0 88.30 0 1205436 0 30355 123427 4.066117608302 1365124.0 1258600.0 24143.0 53164.0 1043.0 1788.0 0.0 103693.0 1205436.0 92.2 1.8 3.9 0.1 0.1 0.0 7.6 88.3 50 50 50.00 38 68256200 27.7 21.4 21.7 29.1 0.0 37.1 23.7 smartseq 1446674 SRR4252098 SRP090061 SRS1699775 SRX2172099 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318317: SK_3_D01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318317 GSM2318317 SK_3_D01_smart-seq 136695200 1366952 2016-09-30 15:56:31 96369150 136695200 1366952 2 1366952 index:0,count:1366952,average:50,stdev:0|index:1,count:1366952,average:50,stdev:0 GSM2318317_r1 5.92 3.08 0.07 117972366 157810883 110702108 149993523 133.77 135.49 1256153 1008277 243.910 2442.035 89 4351 77.08 82.31 1484361 968296 1484361 968296 77.71 78.79 1484361 976170 1484361 926838 12247706 10.38 1.96 0 5.84 0 0.09 0 0.09 0 0.00 0 7.93 0 1256153 0 100 0 98.49 0 1.34 0 0.01 0 1.20 0 0.01 0 307.56 0 0.29 0 26784 0 1366952 0 79798 0 1170 0 1234 0 0 0 108395 0 90 0 0 0 983 0 145454 0 948 0 147475 0 86.06 0 1176355 0 38782 148460 3.828064566036 1366952.0 1256153.0 26784.0 79798.0 1170.0 1234.0 0.0 108395.0 1176355.0 91.9 2.0 5.8 0.1 0.1 0.0 7.9 86.1 50 50 50.00 38 68347600 26.9 22.4 22.7 28.1 0.0 37.3 24.5 smartseq 1446707 SRR4251099 SRP090061 SRS1698777 SRX2171100 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317318: 26Dp4_H11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317318 GSM2317318 26Dp4_H11_smart-seq 117320000 1173200 2016-09-30 15:56:31 82284798 117320000 1173200 2 1173200 index:0,count:1173200,average:50,stdev:0|index:1,count:1173200,average:50,stdev:0 GSM2317318_r1 2.35 3.16 0.22 102061894 127407950 96980134 121968112 124.83 125.77 1077005 889458 275.962 2618.038 110 3105 65.48 69.02 1219310 705251 1219310 705251 65.6 66.09 1219310 706464 1219310 675282 18529868 18.16 1.94 0 4.70 0 0.11 0 0.13 0 0.00 0 7.96 0 1077005 0 100 0 98.68 0 1.41 0 0.01 0 1.20 0 0.01 0 234.64 0 0.27 0 22800 0 1173200 0 55190 0 1270 0 1527 0 0 0 93398 0 60 0 0 0 813 0 99888 0 1034 0 101795 0 87.10 0 1021815 0 33022 103272 3.127369632366 1173200.0 1077005.0 22800.0 55190.0 1270.0 1527.0 0.0 93398.0 1021815.0 91.8 1.9 4.7 0.1 0.1 0.0 8.0 87.1 50 50 50.00 38 58660000 27.6 21.6 21.7 29.1 0.0 37.1 23.4 smartseq 1446708 SRR4252099 SRP090061 SRS1699776 SRX2172100 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318318: SK_3_D02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318318 GSM2318318 SK_3_D02_smart-seq 156271300 1562713 2016-09-30 15:56:31 109466903 156271300 1562713 2 1562713 index:0,count:1562713,average:50,stdev:0|index:1,count:1562713,average:50,stdev:0 GSM2318318_r1 8.62 2.84 0.07 133137900 177301538 123848216 166690592 133.17 134.59 1434668 1194997 222.432 1946.783 78 5578 75.57 81.45 1698322 1084143 1698322 1084143 77.37 78.1 1698322 1109965 1698322 1039607 12769974 9.59 2.09 0 6.63 0 0.10 0 0.08 0 0.00 0 8.02 0 1434668 0 100 0 98.46 0 1.34 0 0.01 0 1.17 0 0.01 0 351.61 0 0.28 0 32708 0 1562713 0 103627 0 1520 0 1202 0 0 0 125323 0 100 0 0 0 1083 0 152223 0 1153 0 154559 0 85.18 0 1331041 0 32839 156752 4.773348762143 1562713.0 1434668.0 32708.0 103627.0 1520.0 1202.0 0.0 125323.0 1331041.0 91.8 2.1 6.6 0.1 0.1 0.0 8.0 85.2 50 50 50.00 38 78135650 26.8 22.5 22.6 28.2 0.0 37.4 24.4 smartseq 1450000 SRR4252100 SRP090061 SRS1699779 SRX2172101 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318319: SK_3_D03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318319 GSM2318319 SK_3_D03_smart-seq 66362100 663621 2016-09-30 15:56:31 47271262 66362100 663621 2 663621 index:0,count:663621,average:50,stdev:0|index:1,count:663621,average:50,stdev:0 GSM2318319_r1 16.19 2.21 0.08 57091148 79724676 53170001 75880993 139.64 142.71 607912 514854 259.946 2303.669 81 2011 74.77 80.56 766227 454515 766227 454515 75.27 77.36 766227 457569 766227 436434 6283744 11.01 1.92 0 6.59 0 0.11 0 0.07 0 0.00 0 8.21 0 607912 0 100 0 98.45 0 1.31 0 0.01 0 1.20 0 0.01 0 125.74 0 0.34 0 12771 0 663621 0 43731 0 756 0 462 0 0 0 54491 0 31 0 0 0 356 0 49563 0 419 0 50369 0 85.02 0 564181 0 24056 49343 2.051172264716 663621.0 607912.0 12771.0 43731.0 756.0 462.0 0.0 54491.0 564181.0 91.6 1.9 6.6 0.1 0.1 0.0 8.2 85.0 50 50 50.00 38 33181050 26.9 22.3 22.5 28.3 0.0 37.2 23.9 smartseq 1450032 SRR4251101 SRP090061 SRS1698778 SRX2171102 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317320: 26Dn4_A02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317320 GSM2317320 26Dn4_A02_smart-seq 133932800 1339328 2016-09-30 15:56:31 91061985 133932800 1339328 2 1339328 index:0,count:1339328,average:50,stdev:0|index:1,count:1339328,average:50,stdev:0 GSM2317320_r1 9.8 0.23 0.01 92592631 103770278 82387111 97749651 112.07 118.65 1016179 1001973 176.807 416.507 105 6172 87.08 98.14 1638056 884912 1638056 884912 88.96 97.28 1638056 903943 1638056 877201 819604 0.89 1.41 0 8.55 0 0.07 0 0.02 0 0.00 0 24.04 0 1016179 0 100 0 98.13 0 1.25 0 0.01 0 1.14 0 0.01 0 301.35 0 0.21 0 18922 0 1339328 0 114467 0 909 0 231 0 0 0 322009 0 1 0 0 0 74 0 10104 0 138 0 10317 0 67.33 0 901712 0 727 10339 14.221458046768 1339328.0 1016179.0 18922.0 114467.0 909.0 231.0 0.0 322009.0 901712.0 75.9 1.4 8.5 0.1 0.0 0.0 24.0 67.3 50 50 50.00 38 66966400 25.7 22.5 22.6 29.2 0.0 37.3 20.9 smartseq 1450033 SRR4252101 SRP090061 SRS1699785 SRX2172102 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318320: SK_3_D04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318320 GSM2318320 SK_3_D04_smart-seq 50602200 506022 2016-09-30 15:56:31 35206640 50602200 506022 2 506022 index:0,count:506022,average:50,stdev:0|index:1,count:506022,average:50,stdev:0 GSM2318320_r1 0.02 0.11 0.01 28759787 28626216 28660436 28568709 99.54 99.68 323974 323044 158.502 447.351 105 2289 98.23 98.67 329290 318246 329290 318246 98.27 98.67 329290 318366 329290 318236 152431 0.53 1.28 0 0.29 0 0.02 0 0.01 0 0.00 0 35.95 0 323974 0 100 0 97.87 0 1.47 0 0.01 0 1.16 0 0.01 0 121.45 0 0.19 0 6469 0 506022 0 1443 0 106 0 52 0 0 0 181890 0 0 0 0 0 11 0 36 0 58 0 105 0 63.74 0 322531 0 25 36 1.440000000000 506022.0 323974.0 6469.0 1443.0 106.0 52.0 0.0 181890.0 322531.0 64.0 1.3 0.3 0.0 0.0 0.0 35.9 63.7 50 50 50.00 38 25301100 25.3 21.1 20.7 32.8 0.0 36.3 17.8 smartseq 1450064 SRR4251102 SRP090061 SRS1698779 SRX2171103 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317321: 26Dn4_A03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317321 GSM2317321 26Dn4_A03_smart-seq 62331800 623318 2016-09-30 15:56:31 43352121 62331800 623318 2 623318 index:0,count:623318,average:50,stdev:0|index:1,count:623318,average:50,stdev:0 GSM2317321_r1 0.83 3.51 0.21 46122079 56908535 44175789 54757405 123.39 123.95 482639 386420 284.869 2806.100 105 1338 70.31 73.5 532677 339321 532677 339321 70.35 70.76 532677 339531 532677 326674 7234911 15.69 1.53 0 3.36 0 0.08 0 0.08 0 0.00 0 22.41 0 482639 0 100 0 98.84 0 1.42 0 0.01 0 1.21 0 0.01 0 140.25 0 0.28 0 9544 0 623318 0 20952 0 523 0 482 0 0 0 139674 0 39 0 0 0 425 0 51432 0 360 0 52256 0 74.07 0 461687 0 21548 52913 2.455587525524 623318.0 482639.0 9544.0 20952.0 523.0 482.0 0.0 139674.0 461687.0 77.4 1.5 3.4 0.1 0.1 0.0 22.4 74.1 50 50 50.00 38 31165900 26.7 22.0 22.4 28.8 0.0 37.3 22.3 smartseq 1450065 SRR4252102 SRP090061 SRS1699777 SRX2172103 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318321: SK_3_D05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318321 GSM2318321 SK_3_D05_smart-seq 117737000 1177370 2016-09-30 15:56:31 82786300 117737000 1177370 2 1177370 index:0,count:1177370,average:50,stdev:0|index:1,count:1177370,average:50,stdev:0 GSM2318321_r1 4.06 3.08 0.1 100214368 126668396 92907320 119442491 126.4 128.56 1071034 884899 248.363 2230.078 81 3594 67.25 72.72 1321175 720262 1321175 720262 68.59 69.57 1321175 734572 1321175 689003 15328819 15.30 2.06 0 6.85 0 0.12 0 0.10 0 0.00 0 8.81 0 1071034 0 100 0 98.54 0 1.36 0 0.01 0 1.19 0 0.01 0 249.33 0 0.29 0 24248 0 1177370 0 80621 0 1431 0 1233 0 0 0 103672 0 97 0 0 0 852 0 108601 0 822 0 110372 0 84.12 0 990413 0 31709 111217 3.507426913495 1177370.0 1071034.0 24248.0 80621.0 1431.0 1233.0 0.0 103672.0 990413.0 91.0 2.1 6.8 0.1 0.1 0.0 8.8 84.1 50 50 50.00 38 58868500 26.9 22.3 22.4 28.5 0.0 37.3 24.0 smartseq 1450096 SRR4252103 SRP090061 SRS1699778 SRX2172104 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318322: SK_3_D06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318322 GSM2318322 SK_3_D06_smart-seq 118525700 1185257 2016-09-30 15:56:31 83276868 118525700 1185257 2 1185257 index:0,count:1185257,average:50,stdev:0|index:1,count:1185257,average:50,stdev:0 GSM2318322_r1 1.45 3.2 0.16 100591964 126047674 93729866 119059427 125.31 127.02 1076679 907082 237.769 1908.739 78 3788 67.22 72.28 1302052 723690 1302052 723690 68.25 69.1 1302052 734872 1302052 691902 15897840 15.80 2.00 0 6.36 0 0.11 0 0.12 0 0.00 0 8.93 0 1076679 0 100 0 98.55 0 1.41 0 0.01 0 1.20 0 0.01 0 284.46 0 0.27 0 23719 0 1185257 0 75396 0 1308 0 1460 0 0 0 105810 0 78 0 0 0 790 0 101608 0 830 0 103306 0 84.48 0 1001283 0 25937 104788 4.040097158499 1185257.0 1076679.0 23719.0 75396.0 1308.0 1460.0 0.0 105810.0 1001283.0 90.8 2.0 6.4 0.1 0.1 0.0 8.9 84.5 50 50 50.00 38 59262850 27.1 21.9 22.0 29.0 0.0 37.3 23.9 smartseq 1450130 SRR4251104 SRP090061 SRS1698781 SRX2171105 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317323: 26Dn4_A05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317323 GSM2317323 26Dn4_A05_smart-seq 174463300 1744633 2016-09-30 15:56:31 118979116 174463300 1744633 2 1744633 index:0,count:1744633,average:50,stdev:0|index:1,count:1744633,average:50,stdev:0 GSM2317323_r1 2.21 3.3 0.12 149183714 183666359 140779400 174622356 123.11 124.04 1581302 1300024 253.214 2206.490 100 5071 68.47 72.69 1832094 1082757 1832094 1082757 68.77 69.5 1832094 1087534 1832094 1035260 23594363 15.82 1.95 0 5.26 0 0.13 0 0.10 0 0.00 0 9.14 0 1581302 0 100 0 98.63 0 1.44 0 0.01 0 1.24 0 0.01 0 224.31 0 0.25 0 34021 0 1744633 0 91826 0 2202 0 1722 0 0 0 159407 0 134 0 0 0 1260 0 163360 0 1187 0 165941 0 85.37 0 1489476 0 30220 169458 5.607478491066 1744633.0 1581302.0 34021.0 91826.0 2202.0 1722.0 0.0 159407.0 1489476.0 90.6 2.0 5.3 0.1 0.1 0.0 9.1 85.4 50 50 50.00 38 87231650 26.9 22.2 22.4 28.5 0.0 37.5 23.8 smartseq 1450131 SRR4252104 SRP090061 SRS1699781 SRX2172105 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318323: SK_3_D07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318323 GSM2318323 SK_3_D07_smart-seq 156517800 1565178 2016-09-30 15:56:31 109478505 156517800 1565178 2 1565178 index:0,count:1565178,average:50,stdev:0|index:1,count:1565178,average:50,stdev:0 GSM2318323_r1 17.87 1.81 0.07 121668481 175416602 103336184 155584639 144.18 150.56 1343299 1217177 196.505 928.057 69 6791 83.22 98.57 1996084 1117879 1996084 1117879 90.57 95.4 1996084 1216638 1996084 1081924 691747 0.57 3.02 0 13.37 0 0.09 0 0.01 0 0.00 0 14.08 0 1343299 0 100 0 97.95 0 1.18 0 0.01 0 1.13 0 0.00 0 216.72 0 0.30 0 47299 0 1565178 0 209194 0 1429 0 120 0 0 0 220330 0 53 0 0 0 483 0 99524 0 546 0 100606 0 72.46 0 1134105 0 8112 102600 12.647928994083 1565178.0 1343299.0 47299.0 209194.0 1429.0 120.0 0.0 220330.0 1134105.0 85.8 3.0 13.4 0.1 0.0 0.0 14.1 72.5 50 50 50.00 38 78258900 25.6 23.1 23.1 28.2 0.0 37.2 22.5 smartseq 1450161 SRR4251105 SRP090061 SRS1698784 SRX2171106 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317324: 26Dn4_A07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317324 GSM2317324 26Dn4_A07_smart-seq 169988300 1699883 2016-09-30 15:56:31 116480079 169988300 1699883 2 1699883 index:0,count:1699883,average:50,stdev:0|index:1,count:1699883,average:50,stdev:0 GSM2317324_r1 5.6 3.23 0.08 146868643 185499024 138234057 176267288 126.3 127.51 1549558 1283636 260.362 2303.266 100 4746 68.65 73.07 1796210 1063737 1796210 1063737 69.44 70.07 1796210 1075969 1796210 1020126 22514481 15.33 1.94 0 5.51 0 0.09 0 0.09 0 0.00 0 8.67 0 1549558 0 100 0 98.67 0 1.32 0 0.01 0 1.22 0 0.01 0 278.16 0 0.26 0 33045 0 1699883 0 93748 0 1484 0 1537 0 0 0 147304 0 111 0 0 0 1123 0 152606 0 1260 0 155100 0 85.64 0 1455810 0 30188 159779 5.292798462965 1699883.0 1549558.0 33045.0 93748.0 1484.0 1537.0 0.0 147304.0 1455810.0 91.2 1.9 5.5 0.1 0.1 0.0 8.7 85.6 50 50 50.00 38 84994150 27.0 22.2 22.3 28.5 0.0 37.5 23.9 smartseq 1450162 SRR4252105 SRP090061 SRS1699782 SRX2172106 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318324: SK_3_D08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318324 GSM2318324 SK_3_D08_smart-seq 114709300 1147093 2016-09-30 15:56:31 81037628 114709300 1147093 2 1147093 index:0,count:1147093,average:50,stdev:0|index:1,count:1147093,average:50,stdev:0 GSM2318324_r1 3.49 3.22 0.06 98109705 129278413 93175708 123980199 131.77 133.06 1046918 823366 248.761 2849.882 81 3498 79.3 83.65 1202490 830193 1202490 830193 79.08 80.26 1202490 827912 1202490 796505 9021290 9.20 1.77 0 4.75 0 0.08 0 0.07 0 0.00 0 8.59 0 1046918 0 100 0 98.53 0 1.38 0 0.01 0 1.23 0 0.01 0 242.91 0 0.29 0 20299 0 1147093 0 54456 0 870 0 752 0 0 0 98553 0 108 0 0 0 854 0 131039 0 754 0 132755 0 86.52 0 992462 0 35214 132540 3.763843925711 1147093.0 1046918.0 20299.0 54456.0 870.0 752.0 0.0 98553.0 992462.0 91.3 1.8 4.7 0.1 0.1 0.0 8.6 86.5 50 50 50.00 38 57354650 26.7 22.6 22.7 28.0 0.0 37.3 24.4 smartseq 1450224 SRR4252107 SRP090061 SRS1699783 SRX2172108 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318326: SK_3_D10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318326 GSM2318326 SK_3_D10_smart-seq 12230600 122306 2016-09-30 15:56:31 8676118 12230600 122306 2 122306 index:0,count:122306,average:50,stdev:0|index:1,count:122306,average:50,stdev:0 GSM2318326_r1 6.89 2.58 0.05 5371619 7744185 4525765 6966888 144.17 153.94 68315 63224 115.797 2170.126 56 902 71.15 84.72 115507 48608 115507 48608 73.54 80.55 115507 50238 115507 46219 340705 6.34 3.13 0 8.94 0 0.38 0 0.10 0 0.00 0 43.66 0 68315 0 100 0 94.23 0 1.27 0 0.01 0 1.10 0 0.01 0 33.87 0 0.45 0 3827 0 122306 0 10939 0 466 0 122 0 0 0 53403 0 8 0 0 0 22 0 6425 0 91 0 6546 0 46.91 0 57376 0 1564 6228 3.982097186701 122306.0 68315.0 3827.0 10939.0 466.0 122.0 0.0 53403.0 57376.0 55.9 3.1 8.9 0.4 0.1 0.0 43.7 46.9 50 50 50.00 38 6115300 22.8 21.0 20.5 35.7 0.0 35.4 16.0 smartseq 1450256 SRR4252108 SRP090061 SRS1699784 SRX2172109 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318327: SK_3_D11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318327 GSM2318327 SK_3_D11_smart-seq 177765500 1777655 2016-09-30 15:56:31 123756261 177765500 1777655 2 1777655 index:0,count:1777655,average:50,stdev:0|index:1,count:1777655,average:50,stdev:0 GSM2318327_r1 8.76 2.36 0.07 147950881 204235873 129969132 186115514 138.04 143.2 1612781 1376489 198.750 1706.860 83 7610 75.99 86.79 2300992 1225502 2300992 1225502 78.83 82.37 2300992 1271351 2300992 1163124 9638109 6.51 2.50 0 11.29 0 0.09 0 0.06 0 0.00 0 9.12 0 1612781 0 100 0 98.09 0 1.24 0 0.01 0 1.16 0 0.01 0 237.02 0 0.29 0 44357 0 1777655 0 200687 0 1668 0 1071 0 0 0 162135 0 163 0 0 0 1142 0 176183 0 1235 0 178723 0 79.44 0 1412094 0 23126 181701 7.857000778345 1777655.0 1612781.0 44357.0 200687.0 1668.0 1071.0 0.0 162135.0 1412094.0 90.7 2.5 11.3 0.1 0.1 0.0 9.1 79.4 50 50 50.00 38 88882750 25.9 23.1 23.3 27.6 0.0 37.4 23.8 smartseq 1450512 SRR4252110 SRP090061 SRS1699787 SRX2172111 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318329: SK_3_E01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318329 GSM2318329 SK_3_E01_smart-seq 241956500 2419565 2016-09-30 15:56:31 167124799 241956500 2419565 2 2419565 index:0,count:2419565,average:50,stdev:0|index:1,count:2419565,average:50,stdev:0 GSM2318329_r1 3.0 3.17 0.13 209727080 270160963 194221114 253867798 128.82 130.71 2243262 1829966 226.923 2168.877 83 8442 72.64 78.59 2738789 1629481 2738789 1629481 73.93 75.11 2738789 1658457 2738789 1557282 24922114 11.88 1.99 0 7.02 0 0.09 0 0.09 0 0.00 0 7.10 0 2243262 0 100 0 98.53 0 1.37 0 0.01 0 1.18 0 0.01 0 362.93 0 0.25 0 48105 0 2419565 0 169883 0 2290 0 2123 0 0 0 171890 0 214 0 0 0 2047 0 263065 0 1733 0 267059 0 85.69 0 2073379 0 40907 271746 6.643019532109 2419565.0 2243262.0 48105.0 169883.0 2290.0 2123.0 0.0 171890.0 2073379.0 92.7 2.0 7.0 0.1 0.1 0.0 7.1 85.7 50 50 50.00 38 120978250 26.7 22.6 22.8 27.9 0.0 37.4 24.6 smartseq 1450544 SRR4251111 SRP090061 SRS1698789 SRX2171112 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317330: 26Dn4_B02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317330 GSM2317330 26Dn4_B02_smart-seq 198849900 1988499 2016-09-30 15:56:31 135188593 198849900 1988499 2 1988499 index:0,count:1988499,average:50,stdev:0|index:1,count:1988499,average:50,stdev:0 GSM2317330_r1 5.13 3.11 0.17 173616725 216644754 164243020 206516980 124.78 125.74 1826465 1507267 260.188 2326.365 105 5575 67.34 71.3 2082427 1229953 2082427 1229953 68.2 68.57 2082427 1245669 2082427 1182814 27292302 15.72 1.93 0 5.10 0 0.09 0 0.11 0 0.00 0 7.94 0 1826465 0 100 0 98.75 0 1.33 0 0.01 0 1.20 0 0.01 0 275.33 0 0.25 0 38416 0 1988499 0 101367 0 1796 0 2252 0 0 0 157986 0 179 0 0 0 1383 0 182821 0 1389 0 185772 0 86.75 0 1725098 0 32280 190393 5.898172242875 1988499.0 1826465.0 38416.0 101367.0 1796.0 2252.0 0.0 157986.0 1725098.0 91.9 1.9 5.1 0.1 0.1 0.0 7.9 86.8 50 50 50.00 38 99424950 27.0 22.2 22.4 28.4 0.0 37.6 24.1 smartseq 1450545 SRR4252111 SRP090061 SRS1699788 SRX2172112 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318330: SK_3_E02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318330 GSM2318330 SK_3_E02_smart-seq 119072000 1190720 2016-09-30 15:56:31 82008453 119072000 1190720 2 1190720 index:0,count:1190720,average:50,stdev:0|index:1,count:1190720,average:50,stdev:0 GSM2318330_r1 1.73 0.77 0.06 83404526 88962421 73944028 83727815 106.66 113.23 942440 913863 152.280 743.129 83 6404 65.63 74.08 1544215 618541 1544215 618541 66.94 73.15 1544215 630876 1544215 610775 8569955 10.28 2.18 0 9.02 0 0.29 0 0.09 0 0.00 0 20.47 0 942440 0 100 0 97.87 0 1.47 0 0.01 0 1.14 0 0.01 0 214.33 0 0.23 0 26002 0 1190720 0 107448 0 3448 0 1085 0 0 0 243747 0 39 0 0 0 196 0 23513 0 604 0 24352 0 70.12 0 834992 0 2767 24758 8.947596675099 1190720.0 942440.0 26002.0 107448.0 3448.0 1085.0 0.0 243747.0 834992.0 79.1 2.2 9.0 0.3 0.1 0.0 20.5 70.1 50 50 50.00 38 59536000 26.1 21.9 21.9 30.1 0.0 37.0 20.8 smartseq 1450576 SRR4252112 SRP090061 SRS1699790 SRX2172113 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318331: SK_3_E03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318331 GSM2318331 SK_3_E03_smart-seq 211751600 2117516 2016-09-30 15:56:31 147198314 211751600 2117516 2 2117516 index:0,count:2117516,average:50,stdev:0|index:1,count:2117516,average:50,stdev:0 GSM2318331_r1 7.11 2.27 0.09 176084852 239129579 161763644 222708672 135.8 137.68 1895973 1533922 224.486 2000.688 83 7313 86.36 94.27 2301612 1637338 2301612 1637338 88.44 90.12 2301612 1676747 2301612 1565262 4228100 2.40 2.21 0 7.51 0 0.07 0 0.03 0 0.00 0 10.36 0 1895973 0 100 0 98.35 0 1.34 0 0.01 0 1.20 0 0.01 0 346.50 0 0.25 0 46821 0 2117516 0 159117 0 1577 0 571 0 0 0 219395 0 169 0 0 0 1808 0 239370 0 1127 0 242474 0 82.02 0 1736856 0 29419 245787 8.354702743125 2117516.0 1895973.0 46821.0 159117.0 1577.0 571.0 0.0 219395.0 1736856.0 89.5 2.2 7.5 0.1 0.0 0.0 10.4 82.0 50 50 50.00 38 105875800 26.2 22.8 22.9 28.1 0.0 37.3 23.4 smartseq 1450608 SRR4252113 SRP090061 SRS1699793 SRX2172114 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318332: SK_3_E04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318332 GSM2318332 SK_3_E04_smart-seq 135741900 1357419 2016-09-30 15:56:31 95078244 135741900 1357419 2 1357419 index:0,count:1357419,average:50,stdev:0|index:1,count:1357419,average:50,stdev:0 GSM2318332_r1 9.13 2.52 0.11 117586211 157100598 110984864 149120446 133.6 134.36 1251274 976029 247.015 2503.327 80 4051 79.88 84.82 1421259 999497 1421259 999497 80.72 81.45 1421259 1010044 1421259 959722 9995488 8.50 2.03 0 5.37 0 0.06 0 0.05 0 0.00 0 7.70 0 1251274 0 100 0 98.49 0 1.30 0 0.01 0 1.20 0 0.01 0 244.34 0 0.26 0 27493 0 1357419 0 72919 0 857 0 713 0 0 0 104575 0 97 0 0 0 1237 0 161100 0 939 0 163373 0 86.81 0 1178355 0 38495 164771 4.280322119756 1357419.0 1251274.0 27493.0 72919.0 857.0 713.0 0.0 104575.0 1178355.0 92.2 2.0 5.4 0.1 0.1 0.0 7.7 86.8 50 50 50.00 38 67870950 26.9 22.3 22.5 28.3 0.0 37.1 23.8 smartseq 1450640 SRR4251114 SRP090061 SRS1698793 SRX2171115 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317333: 26Dn4_B05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317333 GSM2317333 26Dn4_B05_smart-seq 164139800 1641398 2016-09-30 15:56:31 111065475 164139800 1641398 2 1641398 index:0,count:1641398,average:50,stdev:0|index:1,count:1641398,average:50,stdev:0 GSM2317333_r1 6.0 2.79 0.1 143364725 185956944 134693109 176337966 129.71 130.92 1520010 1217553 243.924 2116.589 100 5106 75.48 80.49 1765264 1147333 1765264 1147333 76.07 77.03 1765264 1156254 1765264 1097986 15679471 10.94 1.99 0 5.77 0 0.09 0 0.09 0 0.00 0 7.22 0 1520010 0 100 0 98.50 0 1.35 0 0.01 0 1.20 0 0.01 0 295.45 0 0.25 0 32645 0 1641398 0 94630 0 1526 0 1414 0 0 0 118448 0 126 0 0 0 1372 0 179117 0 1128 0 181743 0 86.84 0 1425380 0 31078 184653 5.941598558466 1641398.0 1520010.0 32645.0 94630.0 1526.0 1414.0 0.0 118448.0 1425380.0 92.6 2.0 5.8 0.1 0.1 0.0 7.2 86.8 50 50 50.00 38 82069900 26.4 23.0 23.2 27.4 0.0 37.6 24.6 smartseq 1450641 SRR4252114 SRP090061 SRS1699792 SRX2172115 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318333: SK_3_E05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318333 GSM2318333 SK_3_E05_smart-seq 176812500 1768125 2016-09-30 15:56:31 122221250 176812500 1768125 2 1768125 index:0,count:1768125,average:50,stdev:0|index:1,count:1768125,average:50,stdev:0 GSM2318333_r1 1.1 2.43 0.08 139643789 172317852 131606808 163953876 123.4 124.58 1514660 1350072 209.533 1415.848 80 6528 67.56 71.83 1808504 1023281 1808504 1023281 68.61 69.52 1808504 1039134 1808504 990404 22930692 16.42 2.17 0 5.09 0 0.09 0 0.10 0 0.00 0 14.14 0 1514660 0 100 0 98.43 0 1.44 0 0.01 0 1.25 0 0.01 0 254.61 0 0.24 0 38319 0 1768125 0 89999 0 1648 0 1784 0 0 0 250033 0 90 0 0 0 809 0 107944 0 1065 0 109908 0 80.57 0 1424661 0 14737 111358 7.556354753342 1768125.0 1514660.0 38319.0 89999.0 1648.0 1784.0 0.0 250033.0 1424661.0 85.7 2.2 5.1 0.1 0.1 0.0 14.1 80.6 50 50 50.00 38 88406250 26.9 21.6 21.6 29.8 0.0 37.2 22.2 smartseq 1450672 SRR4251115 SRP090061 SRS1698792 SRX2171116 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317334: 26Dn4_B06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317334 GSM2317334 26Dn4_B06_smart-seq 130380800 1303808 2016-09-30 15:56:31 89543023 130380800 1303808 2 1303808 index:0,count:1303808,average:50,stdev:0|index:1,count:1303808,average:50,stdev:0 GSM2317334_r1 4.11 3.28 0.19 113444210 145487403 107288463 138447424 128.25 129.04 1194321 936776 279.816 2574.995 101 3317 74.04 78.43 1359182 884287 1359182 884287 74.34 75.13 1359182 887835 1359182 847168 14166834 12.49 2.02 0 5.12 0 0.07 0 0.08 0 0.00 0 8.24 0 1194321 0 100 0 98.60 0 1.39 0 0.01 0 1.22 0 0.01 0 223.51 0 0.26 0 26362 0 1303808 0 66765 0 930 0 1064 0 0 0 107493 0 97 0 0 0 1170 0 142786 0 884 0 144937 0 86.48 0 1127556 0 36399 146891 4.035577900492 1303808.0 1194321.0 26362.0 66765.0 930.0 1064.0 0.0 107493.0 1127556.0 91.6 2.0 5.1 0.1 0.1 0.0 8.2 86.5 50 50 50.00 38 65190400 26.9 22.4 22.5 28.2 0.0 37.4 23.8 smartseq 1450673 SRR4252115 SRP090061 SRS1699791 SRX2172116 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318334: SK_3_E06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318334 GSM2318334 SK_3_E06_smart-seq 174640100 1746401 2016-09-30 15:56:31 120795328 174640100 1746401 2 1746401 index:0,count:1746401,average:50,stdev:0|index:1,count:1746401,average:50,stdev:0 GSM2318334_r1 13.95 2.49 0.07 144716877 203626132 131748611 188786180 140.71 143.29 1565012 1308042 215.068 1872.702 81 6282 89.03 98.14 1962874 1393399 1962874 1393399 91.79 94.27 1962874 1436599 1962874 1338375 753661 0.52 2.28 0 8.32 0 0.07 0 0.03 0 0.00 0 10.29 0 1565012 0 100 0 98.23 0 1.24 0 0.01 0 1.18 0 0.01 0 261.96 0 0.26 0 39735 0 1746401 0 145258 0 1253 0 501 0 0 0 179635 0 119 0 0 0 1272 0 177004 0 725 0 179120 0 81.30 0 1419754 0 24464 181344 7.412688031393 1746401.0 1565012.0 39735.0 145258.0 1253.0 501.0 0.0 179635.0 1419754.0 89.6 2.3 8.3 0.1 0.0 0.0 10.3 81.3 50 50 50.00 38 87320050 26.1 22.8 22.9 28.2 0.0 37.2 22.9 smartseq 1450704 SRR4252116 SRP090061 SRS1699789 SRX2172117 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318335: SK_3_E07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318335 GSM2318335 SK_3_E07_smart-seq 237244000 2372440 2016-09-30 15:56:31 163457715 237244000 2372440 2 2372440 index:0,count:2372440,average:50,stdev:0|index:1,count:2372440,average:50,stdev:0 GSM2318335_r1 0.71 2.96 0.11 197065526 248744183 187980447 238776256 126.22 127.02 2131351 1841109 207.540 1753.119 81 9275 72.81 76.44 2383439 1551818 2383439 1551818 72.5 73.19 2383439 1545187 2383439 1485839 26025495 13.21 1.82 0 4.26 0 0.11 0 0.12 0 0.00 0 9.94 0 2131351 0 100 0 98.55 0 1.39 0 0.01 0 1.21 0 0.01 0 305.03 0 0.23 0 43136 0 2372440 0 101136 0 2496 0 2867 0 0 0 235726 0 105 0 0 0 1746 0 198780 0 1684 0 202315 0 85.57 0 2030215 0 26122 204715 7.836880790139 2372440.0 2131351.0 43136.0 101136.0 2496.0 2867.0 0.0 235726.0 2030215.0 89.8 1.8 4.3 0.1 0.1 0.0 9.9 85.6 50 50 50.00 38 118622000 26.7 22.2 22.2 28.8 0.0 37.3 23.3 smartseq 1450736 SRR4252117 SRP090061 SRS1699794 SRX2172118 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318336: SK_3_E08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318336 GSM2318336 SK_3_E08_smart-seq 171215000 1712150 2016-09-30 15:56:31 118804338 171215000 1712150 2 1712150 index:0,count:1712150,average:50,stdev:0|index:1,count:1712150,average:50,stdev:0 GSM2318336_r1 9.06 2.81 0.08 146903714 199453762 136231526 186647508 135.77 137.01 1574799 1276995 235.780 2242.464 81 5755 79.45 85.91 1852299 1251154 1852299 1251154 81.27 82.13 1852299 1279858 1852299 1196066 11319995 7.71 2.20 0 6.92 0 0.09 0 0.06 0 0.00 0 7.87 0 1574799 0 100 0 98.47 0 1.29 0 0.01 0 1.22 0 0.01 0 237.07 0 0.25 0 37649 0 1712150 0 118493 0 1573 0 1068 0 0 0 134710 0 142 0 0 0 1150 0 188517 0 887 0 190696 0 85.06 0 1456306 0 34018 193134 5.677406079135 1712150.0 1574799.0 37649.0 118493.0 1573.0 1068.0 0.0 134710.0 1456306.0 92.0 2.2 6.9 0.1 0.1 0.0 7.9 85.1 50 50 50.00 38 85607500 26.4 22.7 22.8 28.0 0.0 37.3 23.9 smartseq 1450768 SRR4251118 SRP090061 SRS1698794 SRX2171119 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317337: 26Dn4_B09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317337 GSM2317337 26Dn4_B09_smart-seq 132845800 1328458 2016-09-30 15:56:31 90286157 132845800 1328458 2 1328458 index:0,count:1328458,average:50,stdev:0|index:1,count:1328458,average:50,stdev:0 GSM2317337_r1 1.73 3.87 0.1 114637082 145170869 108038994 138094219 126.64 127.82 1211958 961690 263.827 2586.631 92 3661 73.47 78.09 1400357 890443 1400357 890443 73.9 74.67 1400357 895608 1400357 851498 13947890 12.17 2.02 0 5.40 0 0.12 0 0.09 0 0.00 0 8.56 0 1211958 0 100 0 98.65 0 1.39 0 0.01 0 1.23 0 0.01 0 251.71 0 0.25 0 26870 0 1328458 0 71686 0 1628 0 1195 0 0 0 113677 0 89 0 0 0 1059 0 144143 0 986 0 146277 0 85.83 0 1140272 0 32350 148541 4.591684698609 1328458.0 1211958.0 26870.0 71686.0 1628.0 1195.0 0.0 113677.0 1140272.0 91.2 2.0 5.4 0.1 0.1 0.0 8.6 85.8 50 50 50.00 38 66422900 26.4 22.9 23.0 27.7 0.0 37.6 24.1 smartseq 1450769 SRR4252118 SRP090061 SRS1699795 SRX2172119 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318337: SK_3_E09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318337 GSM2318337 SK_3_E09_smart-seq 127850900 1278509 2016-09-30 15:56:31 87815549 127850900 1278509 2 1278509 index:0,count:1278509,average:50,stdev:0|index:1,count:1278509,average:50,stdev:0 GSM2318337_r1 5.35 3.07 0.11 107691662 134977851 99768778 127310492 125.34 127.61 1161986 1011727 224.015 1693.283 81 4449 63.38 68.58 1446785 736416 1446785 736416 64.72 65.66 1446785 751980 1446785 705102 18966844 17.61 1.98 0 6.90 0 0.13 0 0.13 0 0.00 0 8.85 0 1161986 0 100 0 98.46 0 1.37 0 0.01 0 1.21 0 0.01 0 230.13 0 0.25 0 25350 0 1278509 0 88172 0 1716 0 1692 0 0 0 113115 0 79 0 0 0 794 0 93986 0 930 0 95789 0 83.99 0 1073814 0 26534 97138 3.660887917389 1278509.0 1161986.0 25350.0 88172.0 1716.0 1692.0 0.0 113115.0 1073814.0 90.9 2.0 6.9 0.1 0.1 0.0 8.8 84.0 50 50 50.00 38 63925450 26.9 22.2 22.2 28.6 0.0 37.4 24.2 smartseq 1450800 SRR4252119 SRP090061 SRS1699796 SRX2172120 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318338: SK_3_E10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318338 GSM2318338 SK_3_E10_smart-seq 177309000 1773090 2016-09-30 15:56:31 123151931 177309000 1773090 2 1773090 index:0,count:1773090,average:50,stdev:0|index:1,count:1773090,average:50,stdev:0 GSM2318338_r1 4.84 3.0 0.09 155443478 206728129 145789244 195799662 132.99 134.3 1649892 1294365 241.454 2294.668 100 5626 77.78 83.09 1923587 1283349 1923587 1283349 78.55 79.53 1923587 1296056 1923587 1228330 14101359 9.07 1.92 0 5.94 0 0.10 0 0.08 0 0.00 0 6.77 0 1649892 0 100 0 98.56 0 1.34 0 0.01 0 1.20 0 0.01 0 236.41 0 0.26 0 34000 0 1773090 0 105309 0 1851 0 1373 0 0 0 119974 0 140 0 0 0 1548 0 209710 0 1157 0 212555 0 87.11 0 1544583 0 46424 215466 4.641263139755 1773090.0 1649892.0 34000.0 105309.0 1851.0 1373.0 0.0 119974.0 1544583.0 93.1 1.9 5.9 0.1 0.1 0.0 6.8 87.1 50 50 50.00 38 88654500 26.7 22.6 22.8 27.9 0.0 37.3 24.6 smartseq 1451024 SRR4252120 SRP090061 SRS1699798 SRX2172121 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318339: SK_3_E11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318339 GSM2318339 SK_3_E11_smart-seq 228398200 2283982 2016-09-30 15:56:31 157600123 228398200 2283982 2 2283982 index:0,count:2283982,average:50,stdev:0|index:1,count:2283982,average:50,stdev:0 GSM2318339_r1 3.82 3.17 0.11 199246791 262421811 186256095 247298971 131.71 132.77 2125470 1736604 230.123 2245.154 91 8097 76.53 82.02 2482737 1626647 2482737 1626647 77.19 78.07 2482737 1640719 2482737 1548234 20171666 10.12 1.93 0 6.23 0 0.06 0 0.08 0 0.00 0 6.80 0 2125470 0 100 0 98.54 0 1.33 0 0.01 0 1.19 0 0.01 0 304.53 0 0.25 0 44009 0 2283982 0 142322 0 1321 0 1775 0 0 0 155416 0 169 0 0 0 1717 0 248607 0 1733 0 252226 0 86.83 0 1983148 0 37808 258189 6.828951544647 2283982.0 2125470.0 44009.0 142322.0 1321.0 1775.0 0.0 155416.0 1983148.0 93.1 1.9 6.2 0.1 0.1 0.0 6.8 86.8 50 50 50.00 38 114199100 26.5 22.7 22.9 27.8 0.0 37.4 24.4 smartseq 1451152 SRR4252124 SRP090061 SRS1699799 SRX2172125 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318343: SK_3_F03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318343 GSM2318343 SK_3_F03_smart-seq 123892100 1238921 2016-09-30 15:56:31 86030033 123892100 1238921 2 1238921 index:0,count:1238921,average:50,stdev:0|index:1,count:1238921,average:50,stdev:0 GSM2318343_r1 1.02 0.27 0.01 66825063 68288525 66295648 67833368 102.19 102.32 753070 746625 153.138 447.562 105 5627 97.29 98.16 775304 732669 775304 732669 97.37 97.92 775304 733295 775304 730931 651078 0.97 1.20 0 0.54 0 0.01 0 0.01 0 0.00 0 39.20 0 753070 0 100 0 98.01 0 1.41 0 0.01 0 1.15 0 0.01 0 202.73 0 0.18 0 14913 0 1238921 0 6636 0 131 0 87 0 0 0 485633 0 3 0 0 0 46 0 4729 0 70 0 4848 0 60.25 0 746434 0 3682 4439 1.205594785443 1238921.0 753070.0 14913.0 6636.0 131.0 87.0 0.0 485633.0 746434.0 60.8 1.2 0.5 0.0 0.0 0.0 39.2 60.2 50 50 50.00 38 61946050 25.2 22.0 21.7 31.1 0.0 36.6 19.0 smartseq 1451186 SRR4251125 SRP090061 SRS1698803 SRX2171126 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317344: 26Dn4_C05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317344 GSM2317344 26Dn4_C05_smart-seq 159576000 1595760 2016-09-30 15:56:31 108582934 159576000 1595760 2 1595760 index:0,count:1595760,average:50,stdev:0|index:1,count:1595760,average:50,stdev:0 GSM2317344_r1 3.78 3.14 0.15 138617608 171884138 130752563 163645323 124.0 125.16 1465661 1193836 264.833 2480.837 100 4456 69.05 73.35 1698627 1012078 1698627 1012078 69.91 70.48 1698627 1024648 1698627 972507 21300423 15.37 2.12 0 5.38 0 0.08 0 0.11 0 0.00 0 7.96 0 1465661 0 100 0 98.60 0 1.35 0 0.01 0 1.21 0 0.01 0 273.56 0 0.25 0 33878 0 1595760 0 85802 0 1201 0 1833 0 0 0 127065 0 128 0 0 0 1060 0 152396 0 1170 0 154754 0 86.47 0 1379859 0 28558 156367 5.475418446670 1595760.0 1465661.0 33878.0 85802.0 1201.0 1833.0 0.0 127065.0 1379859.0 91.8 2.1 5.4 0.1 0.1 0.0 8.0 86.5 50 50 50.00 38 79788000 27.0 22.1 22.3 28.6 0.0 37.5 23.8 smartseq 1451187 SRR4252125 SRP090061 SRS1699800 SRX2172126 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318344: SK_3_F04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318344 GSM2318344 SK_3_F04_smart-seq 84602100 846021 2016-09-30 15:56:31 58210295 84602100 846021 2 846021 index:0,count:846021,average:50,stdev:0|index:1,count:846021,average:50,stdev:0 GSM2318344_r1 4.46 0.78 0.02 55162315 60329201 51132580 57451675 109.37 112.36 619651 599298 159.858 611.210 105 4149 91.14 98.52 840266 564739 840266 564739 92.65 97.28 840266 574114 840266 557619 286494 0.52 1.71 0 5.49 0 0.05 0 0.01 0 0.00 0 26.69 0 619651 0 100 0 97.86 0 1.31 0 0.01 0 1.10 0 0.01 0 169.20 0 0.20 0 14461 0 846021 0 46449 0 459 0 97 0 0 0 225814 0 3 0 0 0 96 0 17272 0 133 0 17504 0 67.75 0 573202 0 2041 17550 8.598726114650 846021.0 619651.0 14461.0 46449.0 459.0 97.0 0.0 225814.0 573202.0 73.2 1.7 5.5 0.1 0.0 0.0 26.7 67.8 50 50 50.00 38 42301050 25.3 21.7 21.5 31.4 0.0 36.6 18.9 smartseq 1451217 SRR4251126 SRP090061 SRS1698802 SRX2171127 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317345: 26Dn4_C06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317345 GSM2317345 26Dn4_C06_smart-seq 190390500 1903905 2016-09-30 15:56:31 129199842 190390500 1903905 2 1903905 index:0,count:1903905,average:50,stdev:0|index:1,count:1903905,average:50,stdev:0 GSM2317345_r1 2.36 3.82 0.14 163309015 200608023 155499664 192451927 122.84 123.76 1738825 1435552 250.785 2279.876 88 5659 68.87 72.45 1966468 1197467 1966468 1197467 68.8 69.41 1966468 1196241 1966468 1147226 26325284 16.12 2.02 0 4.52 0 0.11 0 0.16 0 0.00 0 8.40 0 1738825 0 100 0 98.60 0 1.37 0 0.01 0 1.21 0 0.01 0 263.62 0 0.24 0 38491 0 1903905 0 86000 0 2106 0 3044 0 0 0 159930 0 152 0 0 0 1313 0 179130 0 1346 0 181941 0 86.81 0 1652825 0 28259 184821 6.540252662868 1903905.0 1738825.0 38491.0 86000.0 2106.0 3044.0 0.0 159930.0 1652825.0 91.3 2.0 4.5 0.1 0.2 0.0 8.4 86.8 50 50 50.00 38 95195250 26.9 22.2 22.3 28.6 0.0 37.6 23.7 smartseq 1451218 SRR4252126 SRP090061 SRS1699804 SRX2172127 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318345: SK_3_F05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318345 GSM2318345 SK_3_F05_smart-seq 189014400 1890144 2016-09-30 15:56:31 130014887 189014400 1890144 2 1890144 index:0,count:1890144,average:50,stdev:0|index:1,count:1890144,average:50,stdev:0 GSM2318345_r1 3.74 3.15 0.12 161010061 206319252 150586747 194699798 128.14 129.29 1722476 1393367 234.761 2217.285 100 6154 77.63 83.17 2018857 1337129 2018857 1337129 78.64 79.6 2018857 1354493 2018857 1279614 15088634 9.37 2.02 0 6.08 0 0.11 0 0.08 0 0.00 0 8.68 0 1722476 0 100 0 98.48 0 1.34 0 0.01 0 1.21 0 0.01 0 234.64 0 0.24 0 38097 0 1890144 0 114861 0 2068 0 1503 0 0 0 164097 0 186 0 0 0 1333 0 204247 0 1065 0 206831 0 85.05 0 1607615 0 32644 209422 6.415329003799 1890144.0 1722476.0 38097.0 114861.0 2068.0 1503.0 0.0 164097.0 1607615.0 91.1 2.0 6.1 0.1 0.1 0.0 8.7 85.1 50 50 50.00 38 94507200 26.7 22.3 22.4 28.6 0.0 37.4 23.6 smartseq 1451249 SRR4251127 SRP090061 SRS1698804 SRX2171128 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317346: 26Dn4_C07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317346 GSM2317346 26Dn4_C07_smart-seq 163559000 1635590 2016-09-30 15:56:31 111740068 163559000 1635590 2 1635590 index:0,count:1635590,average:50,stdev:0|index:1,count:1635590,average:50,stdev:0 GSM2317346_r1 4.46 3.46 0.2 141063538 177615801 133159407 168994805 125.91 126.91 1497745 1207930 266.690 2501.474 81 4505 71.2 75.59 1721973 1066446 1721973 1066446 71.54 72.21 1721973 1071540 1721973 1018710 19470179 13.80 2.10 0 5.31 0 0.13 0 0.09 0 0.00 0 8.21 0 1497745 0 100 0 98.56 0 1.36 0 0.01 0 1.22 0 0.01 0 226.47 0 0.25 0 34341 0 1635590 0 86893 0 2120 0 1411 0 0 0 134314 0 187 0 0 0 1254 0 165848 0 1106 0 168395 0 86.26 0 1410852 0 35247 170561 4.839021760717 1635590.0 1497745.0 34341.0 86893.0 2120.0 1411.0 0.0 134314.0 1410852.0 91.6 2.1 5.3 0.1 0.1 0.0 8.2 86.3 50 50 50.00 38 81779500 26.8 22.4 22.5 28.2 0.0 37.5 24.0 smartseq 1451250 SRR4252127 SRP090061 SRS1699803 SRX2172128 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318346: SK_3_F06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318346 GSM2318346 SK_3_F06_smart-seq 165430900 1654309 2016-09-30 15:56:31 114680603 165430900 1654309 2 1654309 index:0,count:1654309,average:50,stdev:0|index:1,count:1654309,average:50,stdev:0 GSM2318346_r1 4.05 3.26 0.07 142205497 188274258 132629677 177174695 132.4 133.59 1513351 1221564 245.632 2287.110 79 5100 75.4 81.0 1765839 1141054 1765839 1141054 76.8 77.59 1765839 1162306 1765839 1093052 14436235 10.15 2.00 0 6.33 0 0.07 0 0.08 0 0.00 0 8.37 0 1513351 0 100 0 98.54 0 1.34 0 0.01 0 1.22 0 0.01 0 238.22 0 0.26 0 33006 0 1654309 0 104684 0 1204 0 1300 0 0 0 138454 0 178 0 0 0 1191 0 173891 0 1096 0 176356 0 85.15 0 1408667 0 37125 179874 4.845090909091 1654309.0 1513351.0 33006.0 104684.0 1204.0 1300.0 0.0 138454.0 1408667.0 91.5 2.0 6.3 0.1 0.1 0.0 8.4 85.2 50 50 50.00 38 82715450 26.7 22.4 22.5 28.3 0.0 37.3 24.1 smartseq 1451281 SRR4251128 SRP090061 SRS1698805 SRX2171129 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317347: 26Dn4_C08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317347 GSM2317347 26Dn4_C08_smart-seq 157923400 1579234 2016-09-30 15:56:31 108221582 157923400 1579234 2 1579234 index:0,count:1579234,average:50,stdev:0|index:1,count:1579234,average:50,stdev:0 GSM2317347_r1 5.13 3.43 0.3 138533343 175282292 131015651 166984449 126.53 127.45 1446912 1150642 276.953 2636.671 110 4030 71.82 76.03 1646787 1039112 1646787 1039112 72.59 73.14 1646787 1050308 1646787 999600 18234761 13.16 1.94 0 5.08 0 0.10 0 0.12 0 0.00 0 8.16 0 1446912 0 100 0 98.68 0 1.35 0 0.01 0 1.21 0 0.01 0 227.41 0 0.25 0 30715 0 1579234 0 80279 0 1534 0 1915 0 0 0 128873 0 152 0 0 0 1202 0 163015 0 1312 0 165681 0 86.54 0 1366633 0 33725 169849 5.036293550778 1579234.0 1446912.0 30715.0 80279.0 1534.0 1915.0 0.0 128873.0 1366633.0 91.6 1.9 5.1 0.1 0.1 0.0 8.2 86.5 50 50 50.00 38 78961700 27.0 22.3 22.5 28.3 0.0 37.5 24.0 smartseq 1451282 SRR4252128 SRP090061 SRS1699806 SRX2172129 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318347: SK_3_F07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318347 GSM2318347 SK_3_F07_smart-seq 205036700 2050367 2016-09-30 15:56:31 141169449 205036700 2050367 2 2050367 index:0,count:2050367,average:50,stdev:0|index:1,count:2050367,average:50,stdev:0 GSM2318347_r1 0.7 3.14 0.12 175155130 222782354 166038446 212437123 127.19 127.94 1882600 1555353 228.115 2400.251 78 7102 73.99 78.2 2146471 1393017 2146471 1393017 73.7 74.54 2146471 1387494 2146471 1327868 21428686 12.23 1.90 0 4.94 0 0.09 0 0.08 0 0.00 0 8.01 0 1882600 0 100 0 98.56 0 1.43 0 0.01 0 1.23 0 0.01 0 283.90 0 0.24 0 39021 0 2050367 0 101242 0 1873 0 1629 0 0 0 164265 0 116 0 0 0 1630 0 203862 0 1418 0 207026 0 86.88 0 1781358 0 32749 210503 6.427768786833 2050367.0 1882600.0 39021.0 101242.0 1873.0 1629.0 0.0 164265.0 1781358.0 91.8 1.9 4.9 0.1 0.1 0.0 8.0 86.9 50 50 50.00 38 102518350 26.9 22.3 22.3 28.6 0.0 37.4 24.1 smartseq 1451568 SRR4252131 SRP090061 SRS1699808 SRX2172132 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318350: SK_3_F10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318350 GSM2318350 SK_3_F10_smart-seq 237862900 2378629 2016-09-30 15:56:31 162721336 237862900 2378629 2 2378629 index:0,count:2378629,average:50,stdev:0|index:1,count:2378629,average:50,stdev:0 GSM2318350_r1 4.57 3.22 0.17 200889220 252789904 190746698 241755838 125.84 126.74 2179893 1876554 200.367 2052.773 78 9730 71.16 75.09 2472804 1551252 2472804 1551252 71.29 72.04 2472804 1553999 2472804 1488199 27925164 13.90 1.81 0 4.80 0 0.11 0 0.12 0 0.00 0 8.13 0 2179893 0 100 0 98.55 0 1.35 0 0.01 0 1.19 0 0.01 0 267.60 0 0.24 0 42974 0 2378629 0 114057 0 2572 0 2873 0 0 0 193291 0 165 0 0 0 1441 0 204786 0 1521 0 207913 0 86.85 0 2065836 0 31902 213365 6.688138674691 2378629.0 2179893.0 42974.0 114057.0 2572.0 2873.0 0.0 193291.0 2065836.0 91.6 1.8 4.8 0.1 0.1 0.0 8.1 86.8 50 50 50.00 38 118931450 27.0 22.2 22.2 28.5 0.0 37.5 24.2 smartseq 1451600 SRR4252132 SRP090061 SRS1699807 SRX2172133 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318351: SK_3_F11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318351 GSM2318351 SK_3_F11_smart-seq 257511100 2575111 2016-09-30 15:56:31 175895359 257511100 2575111 2 2575111 index:0,count:2575111,average:50,stdev:0|index:1,count:2575111,average:50,stdev:0 GSM2318351_r1 7.69 2.53 0.07 219344651 292990598 203823374 275709747 133.58 135.27 2372753 2013521 207.700 1668.443 81 10537 74.87 80.78 2827102 1776463 2827102 1776463 76.37 77.35 2827102 1812093 2827102 1701085 21848930 9.96 2.07 0 6.74 0 0.11 0 0.09 0 0.00 0 7.65 0 2372753 0 100 0 98.51 0 1.29 0 0.01 0 1.18 0 0.01 0 356.55 0 0.24 0 53264 0 2575111 0 173602 0 2855 0 2387 0 0 0 197116 0 211 0 0 0 2200 0 249310 0 1804 0 253525 0 85.40 0 2199151 0 36928 258207 6.992173960139 2575111.0 2372753.0 53264.0 173602.0 2855.0 2387.0 0.0 197116.0 2199151.0 92.1 2.1 6.7 0.1 0.1 0.0 7.7 85.4 50 50 50.00 38 128755550 26.7 22.6 22.7 28.1 0.0 37.5 24.6 smartseq 1451632 SRR4252133 SRP090061 SRS1699811 SRX2172134 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318352: SK_3_F12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318352 GSM2318352 SK_3_F12_smart-seq 219089700 2190897 2016-09-30 15:56:31 150824938 219089700 2190897 2 2190897 index:0,count:2190897,average:50,stdev:0|index:1,count:2190897,average:50,stdev:0 GSM2318352_r1 5.36 3.06 0.06 189851544 252949996 176455096 237812629 133.24 134.77 2020048 1596967 234.265 2462.834 83 7313 78.46 84.58 2414052 1584974 2414052 1584974 79.62 80.77 2414052 1608306 2414052 1513613 16323508 8.60 1.97 0 6.67 0 0.07 0 0.07 0 0.00 0 7.66 0 2020048 0 100 0 98.52 0 1.34 0 0.01 0 1.20 0 0.01 0 246.48 0 0.26 0 43170 0 2190897 0 146089 0 1452 0 1589 0 0 0 167808 0 167 0 0 0 1665 0 262621 0 1406 0 265859 0 85.53 0 1873959 0 45873 271214 5.912279554422 2190897.0 2020048.0 43170.0 146089.0 1452.0 1589.0 0.0 167808.0 1873959.0 92.2 2.0 6.7 0.1 0.1 0.0 7.7 85.5 50 50 50.00 38 109544850 26.4 22.9 23.1 27.6 0.0 37.5 24.8 smartseq 1451664 SRR4251134 SRP090061 SRS1698811 SRX2171135 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317353: 26Dn4_D03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317353 GSM2317353 26Dn4_D03_smart-seq 180678800 1806788 2016-09-30 15:56:31 125138632 180678800 1806788 2 1806788 index:0,count:1806788,average:50,stdev:0|index:1,count:1806788,average:50,stdev:0 GSM2317353_r1 5.87 3.63 0.12 155647688 199015846 146379725 188902648 127.86 129.05 1652140 1344286 248.627 2475.215 100 5398 74.52 79.4 1914970 1231142 1914970 1231142 75.38 76.28 1914970 1245399 1914970 1182839 17148611 11.02 2.00 0 5.62 0 0.11 0 0.08 0 0.00 0 8.37 0 1652140 0 100 0 98.50 0 1.31 0 0.01 0 1.19 0 0.01 0 309.74 0 0.27 0 36170 0 1806788 0 101573 0 1985 0 1409 0 0 0 151254 0 153 0 0 0 1364 0 180721 0 1451 0 183689 0 85.82 0 1550567 0 33179 188690 5.687030953314 1806788.0 1652140.0 36170.0 101573.0 1985.0 1409.0 0.0 151254.0 1550567.0 91.4 2.0 5.6 0.1 0.1 0.0 8.4 85.8 50 50 50.00 38 90339400 26.9 22.2 22.4 28.4 0.0 37.5 23.9 smartseq 1451665 SRR4252134 SRP090061 SRS1699809 SRX2172135 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318353: SK_3_G01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318353 GSM2318353 SK_3_G01_smart-seq 234443200 2344432 2016-09-30 15:56:31 161777529 234443200 2344432 2 2344432 index:0,count:2344432,average:50,stdev:0|index:1,count:2344432,average:50,stdev:0 GSM2318353_r1 4.16 3.25 0.07 199664446 265046433 185463347 248636486 132.75 134.06 2144536 1742245 229.908 2173.422 80 8024 75.86 81.85 2528260 1626823 2528260 1626823 77.4 78.29 2528260 1659837 2528260 1556055 19132603 9.58 2.07 0 6.69 0 0.08 0 0.08 0 0.00 0 8.36 0 2144536 0 100 0 98.47 0 1.30 0 0.01 0 1.20 0 0.01 0 272.26 0 0.25 0 48426 0 2344432 0 156902 0 1854 0 1958 0 0 0 196084 0 238 0 0 0 1795 0 253815 0 1466 0 257314 0 84.78 0 1987634 0 40907 262460 6.416016818637 2344432.0 2144536.0 48426.0 156902.0 1854.0 1958.0 0.0 196084.0 1987634.0 91.5 2.1 6.7 0.1 0.1 0.0 8.4 84.8 50 50 50.00 38 117221600 26.6 22.7 22.8 27.9 0.0 37.4 24.5 smartseq 1451696 SRR4251135 SRP090061 SRS1698813 SRX2171136 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317354: 26Dn4_D04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317354 GSM2317354 26Dn4_D04_smart-seq 135812800 1358128 2016-09-30 15:56:31 94308990 135812800 1358128 2 1358128 index:0,count:1358128,average:50,stdev:0|index:1,count:1358128,average:50,stdev:0 GSM2317354_r1 1.71 3.44 0.15 119011818 148988305 112233499 141319181 125.19 125.92 1254371 979744 273.031 2565.342 91 3540 71.5 75.93 1437605 896823 1437605 896823 72.07 72.71 1437605 904007 1437605 858696 16728158 14.06 2.03 0 5.40 0 0.11 0 0.12 0 0.00 0 7.41 0 1254371 0 100 0 98.66 0 1.41 0 0.01 0 1.22 0 0.01 0 222.24 0 0.27 0 27537 0 1358128 0 73320 0 1504 0 1678 0 0 0 100575 0 134 0 0 0 1340 0 153442 0 1028 0 155944 0 86.96 0 1181051 0 38155 158670 4.158563753112 1358128.0 1254371.0 27537.0 73320.0 1504.0 1678.0 0.0 100575.0 1181051.0 92.4 2.0 5.4 0.1 0.1 0.0 7.4 87.0 50 50 50.00 38 67906400 27.1 22.2 22.4 28.3 0.0 37.4 23.9 smartseq 1451697 SRR4252135 SRP090061 SRS1699812 SRX2172136 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318354: SK_3_G02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318354 GSM2318354 SK_3_G02_smart-seq 180977100 1809771 2016-09-30 15:56:31 125047991 180977100 1809771 2 1809771 index:0,count:1809771,average:50,stdev:0|index:1,count:1809771,average:50,stdev:0 GSM2318354_r1 4.42 2.93 0.08 154780052 203920245 145200499 193003632 131.75 132.92 1656240 1290955 237.436 2483.101 78 5802 78.76 84.14 1910086 1304440 1910086 1304440 79.17 80.19 1910086 1311247 1910086 1243188 13613104 8.80 2.02 0 5.85 0 0.08 0 0.06 0 0.00 0 8.34 0 1656240 0 100 0 98.46 0 1.38 0 0.01 0 1.20 0 0.01 0 250.58 0 0.26 0 36535 0 1809771 0 105850 0 1482 0 1145 0 0 0 150904 0 164 0 0 0 1608 0 221583 0 1235 0 224590 0 85.67 0 1550390 0 46160 227831 4.935680242634 1809771.0 1656240.0 36535.0 105850.0 1482.0 1145.0 0.0 150904.0 1550390.0 91.5 2.0 5.8 0.1 0.1 0.0 8.3 85.7 50 50 50.00 38 90488550 26.5 22.9 23.0 27.6 0.0 37.4 24.7 smartseq 1451728 SRR4251136 SRP090061 SRS1698814 SRX2171137 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317355: 26Dn4_D05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317355 GSM2317355 26Dn4_D05_smart-seq 144442000 1444420 2016-09-30 15:56:31 99940615 144442000 1444420 2 1444420 index:0,count:1444420,average:50,stdev:0|index:1,count:1444420,average:50,stdev:0 GSM2317355_r1 0.69 3.29 0.11 124798701 154935082 117843701 147155746 124.15 124.87 1317721 1052624 269.916 2499.255 83 3844 68.75 72.92 1504534 905888 1504534 905888 69.2 69.76 1504534 911885 1504534 866609 19085172 15.29 1.99 0 5.23 0 0.10 0 0.09 0 0.00 0 8.58 0 1317721 0 100 0 98.68 0 1.43 0 0.01 0 1.20 0 0.01 0 288.88 0 0.27 0 28745 0 1444420 0 75472 0 1425 0 1318 0 0 0 123956 0 179 0 0 0 1126 0 148643 0 1122 0 151070 0 86.00 0 1242249 0 37326 152452 4.084337994963 1444420.0 1317721.0 28745.0 75472.0 1425.0 1318.0 0.0 123956.0 1242249.0 91.2 2.0 5.2 0.1 0.1 0.0 8.6 86.0 50 50 50.00 38 72221000 27.1 22.2 22.4 28.3 0.0 37.4 23.9 smartseq 1451729 SRR4252136 SRP090061 SRS1699813 SRX2172137 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318355: SK_3_G03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318355 GSM2318355 SK_3_G03_smart-seq 208836900 2088369 2016-09-30 15:56:31 143825147 208836900 2088369 2 2088369 index:0,count:2088369,average:50,stdev:0|index:1,count:2088369,average:50,stdev:0 GSM2318355_r1 15.15 1.63 0.07 149213326 204103713 128768954 182376344 136.79 141.63 1658628 1525973 175.385 819.232 81 9661 84.11 97.92 2403894 1395013 2403894 1395013 90.54 94.93 2403894 1501732 2403894 1352442 1170543 0.78 2.52 0 11.20 0 0.08 0 0.02 0 0.00 0 20.48 0 1658628 0 100 0 97.95 0 1.17 0 0.01 0 1.13 0 0.00 0 268.50 0 0.26 0 52570 0 2088369 0 233917 0 1716 0 369 0 0 0 427656 0 74 0 0 0 854 0 121307 0 603 0 122838 0 68.22 0 1424711 0 7481 124525 16.645501938244 2088369.0 1658628.0 52570.0 233917.0 1716.0 369.0 0.0 427656.0 1424711.0 79.4 2.5 11.2 0.1 0.0 0.0 20.5 68.2 50 50 50.00 38 104418450 25.5 22.9 22.8 28.8 0.0 37.2 21.4 smartseq 1451760 SRR4251137 SRP090061 SRS1698812 SRX2171138 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317356: 26Dn4_D06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317356 GSM2317356 26Dn4_D06_smart-seq 154327900 1543279 2016-09-30 15:56:31 106437503 154327900 1543279 2 1543279 index:0,count:1543279,average:50,stdev:0|index:1,count:1543279,average:50,stdev:0 GSM2317356_r1 1.87 3.32 0.15 133390216 165529627 126512937 158191853 124.09 125.04 1417333 1180565 250.196 2339.175 100 4613 68.44 72.28 1614239 970082 1614239 970082 69.1 69.59 1614239 979365 1614239 933908 21713271 16.28 1.98 0 4.88 0 0.09 0 0.13 0 0.00 0 7.95 0 1417333 0 100 0 98.61 0 1.40 0 0.01 0 1.21 0 0.01 0 213.68 0 0.26 0 30592 0 1543279 0 75262 0 1321 0 2004 0 0 0 122621 0 139 0 0 0 1068 0 134857 0 1114 0 137178 0 86.96 0 1342071 0 27041 138310 5.114825635147 1543279.0 1417333.0 30592.0 75262.0 1321.0 2004.0 0.0 122621.0 1342071.0 91.8 2.0 4.9 0.1 0.1 0.0 7.9 87.0 50 50 50.00 38 77163950 27.2 22.0 22.2 28.7 0.0 37.5 23.9 smartseq 1451761 SRR4252137 SRP090061 SRS1699816 SRX2172138 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318356: SK_3_G04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318356 GSM2318356 SK_3_G04_smart-seq 149535200 1495352 2016-09-30 15:56:31 104759960 149535200 1495352 2 1495352 index:0,count:1495352,average:50,stdev:0|index:1,count:1495352,average:50,stdev:0 GSM2318356_r1 16.17 2.3 0.09 118932828 167266071 109478925 158205571 140.64 144.51 1289252 1127158 203.743 1777.810 81 5702 75.11 81.93 1693916 968421 1693916 968421 75.67 78.33 1693916 975639 1693916 925799 11686729 9.83 1.83 0 7.17 0 0.10 0 0.07 0 0.00 0 13.62 0 1289252 0 100 0 98.35 0 1.26 0 0.01 0 1.17 0 0.01 0 215.33 0 0.28 0 27364 0 1495352 0 107284 0 1458 0 1032 0 0 0 203610 0 55 0 0 0 802 0 107072 0 855 0 108784 0 79.04 0 1181968 0 36712 108023 2.942443887557 1495352.0 1289252.0 27364.0 107284.0 1458.0 1032.0 0.0 203610.0 1181968.0 86.2 1.8 7.2 0.1 0.1 0.0 13.6 79.0 50 50 50.00 38 74767600 26.4 22.2 22.4 29.0 0.0 37.0 21.8 smartseq 1451792 SRR4251138 SRP090061 SRS1698818 SRX2171139 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317357: 26Dn4_D07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317357 GSM2317357 26Dn4_D07_smart-seq 168518800 1685188 2016-09-30 15:56:31 116373063 168518800 1685188 2 1685188 index:0,count:1685188,average:50,stdev:0|index:1,count:1685188,average:50,stdev:0 GSM2317357_r1 1.84 3.26 0.15 144752848 180315805 137138133 171941290 124.57 125.38 1538068 1243852 254.608 2140.266 81 4910 70.99 75.07 1748866 1091899 1748866 1091899 71.1 71.82 1748866 1093508 1748866 1044647 21239967 14.67 2.01 0 4.96 0 0.08 0 0.10 0 0.00 0 8.55 0 1538068 0 100 0 98.59 0 1.45 0 0.01 0 1.21 0 0.01 0 224.69 0 0.26 0 33815 0 1685188 0 83561 0 1405 0 1673 0 0 0 144042 0 162 0 0 0 1510 0 172230 0 1255 0 175157 0 86.31 0 1454507 0 32866 176819 5.379997565874 1685188.0 1538068.0 33815.0 83561.0 1405.0 1673.0 0.0 144042.0 1454507.0 91.3 2.0 5.0 0.1 0.1 0.0 8.5 86.3 50 50 50.00 38 84259400 27.2 22.0 22.2 28.6 0.0 37.5 24.1 smartseq 1451793 SRR4252138 SRP090061 SRS1699814 SRX2172139 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318357: SK_3_G05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318357 GSM2318357 SK_3_G05_smart-seq 246829300 2468293 2016-09-30 15:56:31 169593987 246829300 2468293 2 2468293 index:0,count:2468293,average:50,stdev:0|index:1,count:2468293,average:50,stdev:0 GSM2318357_r1 7.81 3.12 0.08 215443697 289620758 200326249 271865890 134.43 135.71 2305782 1859398 225.049 2182.122 81 8797 78.86 85.01 2691191 1818365 2691191 1818365 80.52 81.33 2691191 1856645 2691191 1739854 17034589 7.91 2.03 0 6.75 0 0.08 0 0.06 0 0.00 0 6.45 0 2305782 0 100 0 98.50 0 1.29 0 0.01 0 1.19 0 0.00 0 286.64 0 0.24 0 50128 0 2468293 0 166656 0 1908 0 1519 0 0 0 159084 0 147 0 0 0 2057 0 287854 0 1689 0 291747 0 86.66 0 2139126 0 47651 296987 6.232544962330 2468293.0 2305782.0 50128.0 166656.0 1908.0 1519.0 0.0 159084.0 2139126.0 93.4 2.0 6.8 0.1 0.1 0.0 6.4 86.7 50 50 50.00 38 123414650 26.6 22.7 22.9 27.7 0.0 37.5 25.0 smartseq 1451824 SRR4252139 SRP090061 SRS1699817 SRX2172140 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318358: SK_3_G06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318358 GSM2318358 SK_3_G06_smart-seq 249151700 2491517 2016-09-30 15:56:31 170388856 249151700 2491517 2 2491517 index:0,count:2491517,average:50,stdev:0|index:1,count:2491517,average:50,stdev:0 GSM2318358_r1 4.39 2.7 0.12 213718561 276932769 200973878 262700400 129.58 130.71 2307904 1959745 205.488 1648.100 81 10208 74.2 79.05 2680236 1712365 2680236 1712365 74.79 75.55 2680236 1726064 2680236 1636642 23845683 11.16 1.97 0 5.69 0 0.09 0 0.07 0 0.00 0 7.21 0 2307904 0 100 0 98.54 0 1.33 0 0.01 0 1.18 0 0.01 0 263.81 0 0.24 0 48961 0 2491517 0 141685 0 2134 0 1753 0 0 0 179726 0 203 0 0 0 1838 0 239732 0 1603 0 243376 0 86.94 0 2166219 0 34551 248266 7.185493907557 2491517.0 2307904.0 48961.0 141685.0 2134.0 1753.0 0.0 179726.0 2166219.0 92.6 2.0 5.7 0.1 0.1 0.0 7.2 86.9 50 50 50.00 38 124575850 26.8 22.4 22.6 28.2 0.0 37.5 24.5 smartseq 1452048 SRR4252140 SRP090061 SRS1699815 SRX2172141 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318359: SK_3_G07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318359 GSM2318359 SK_3_G07_smart-seq 293482700 2934827 2016-09-30 15:56:31 200553633 293482700 2934827 2 2934827 index:0,count:2934827,average:50,stdev:0|index:1,count:2934827,average:50,stdev:0 GSM2318359_r1 2.48 3.68 0.07 249413715 314280925 231474303 295329375 126.01 127.59 2698325 2271166 202.985 1795.571 83 12282 72.14 77.88 3273421 1946627 3273421 1946627 73.2 74.19 3273421 1975045 3273421 1854522 30140873 12.08 2.01 0 6.77 0 0.10 0 0.10 0 0.00 0 7.86 0 2698325 0 100 0 98.48 0 1.34 0 0.01 0 1.19 0 0.01 0 330.17 0 0.23 0 58956 0 2934827 0 198659 0 2976 0 2792 0 0 0 230734 0 339 0 0 0 2211 0 306366 0 1876 0 310792 0 85.17 0 2499666 0 35347 315837 8.935326901859 2934827.0 2698325.0 58956.0 198659.0 2976.0 2792.0 0.0 230734.0 2499666.0 91.9 2.0 6.8 0.1 0.1 0.0 7.9 85.2 50 50 50.00 38 146741350 26.6 22.6 22.8 28.0 0.0 37.5 24.5 smartseq 1452080 SRR4252141 SRP090061 SRS1699819 SRX2172142 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318360: SK_3_G08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318360 GSM2318360 SK_3_G08_smart-seq 185675200 1856752 2016-09-30 15:56:31 128412273 185675200 1856752 2 1856752 index:0,count:1856752,average:50,stdev:0|index:1,count:1856752,average:50,stdev:0 GSM2318360_r1 9.76 3.13 0.08 161024599 225349742 150086414 212253137 139.95 141.42 1716116 1319455 241.936 2600.772 90 5925 84.42 90.8 2018219 1448749 2018219 1448749 85.46 86.83 2018219 1466581 2018219 1385389 7789332 4.84 2.12 0 6.50 0 0.06 0 0.04 0 0.00 0 7.47 0 1716116 0 100 0 98.42 0 1.31 0 0.01 0 1.17 0 0.00 0 230.49 0 0.27 0 39319 0 1856752 0 120656 0 1158 0 827 0 0 0 138651 0 153 0 0 0 1586 0 246277 0 1488 0 249504 0 85.93 0 1595460 0 45539 252149 5.536990272074 1856752.0 1716116.0 39319.0 120656.0 1158.0 827.0 0.0 138651.0 1595460.0 92.4 2.1 6.5 0.1 0.0 0.0 7.5 85.9 50 50 50.00 38 92837600 26.1 23.4 23.6 27.0 0.0 37.4 24.9 smartseq 1452112 SRR4252142 SRP090061 SRS1699820 SRX2172143 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318361: SK_3_G09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318361 GSM2318361 SK_3_G09_smart-seq 126862400 1268624 2016-09-30 15:56:31 87265701 126862400 1268624 2 1268624 index:0,count:1268624,average:50,stdev:0|index:1,count:1268624,average:50,stdev:0 GSM2318361_r1 17.23 2.29 0.08 110041607 156108943 101713421 147977622 141.86 145.48 1177765 1003044 233.504 2153.394 69 4334 76.2 82.75 1520774 897460 1520774 897460 76.73 79.2 1520774 903674 1520774 858934 10407801 9.46 1.92 0 7.35 0 0.10 0 0.07 0 0.00 0 6.99 0 1177765 0 100 0 98.42 0 1.26 0 0.01 0 1.19 0 0.01 0 253.72 0 0.29 0 24306 0 1268624 0 93189 0 1295 0 826 0 0 0 88738 0 76 0 0 0 745 0 100514 0 769 0 102104 0 85.49 0 1084576 0 35594 101699 2.857195032871 1268624.0 1177765.0 24306.0 93189.0 1295.0 826.0 0.0 88738.0 1084576.0 92.8 1.9 7.3 0.1 0.1 0.0 7.0 85.5 50 50 50.00 38 63431200 26.6 22.7 22.9 27.8 0.0 37.4 24.4 smartseq 1452144 SRR4252143 SRP090061 SRS1699818 SRX2172144 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318362: SK_3_G10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318362 GSM2318362 SK_3_G10_smart-seq 201350400 2013504 2016-09-30 15:56:31 138771197 201350400 2013504 2 2013504 index:0,count:2013504,average:50,stdev:0|index:1,count:2013504,average:50,stdev:0 GSM2318362_r1 8.33 2.88 0.11 174159815 235257809 161011245 219767493 135.08 136.49 1864250 1495296 229.010 2076.897 81 7047 78.46 85.09 2213403 1462780 2213403 1462780 80.25 81.28 2213403 1496099 2213403 1397284 14055394 8.07 2.10 0 7.21 0 0.09 0 0.07 0 0.00 0 7.25 0 1864250 0 100 0 98.47 0 1.32 0 0.01 0 1.20 0 0.01 0 249.95 0 0.26 0 42368 0 2013504 0 145104 0 1852 0 1327 0 0 0 146075 0 175 0 0 0 1749 0 237007 0 1202 0 240133 0 85.38 0 1719146 0 43626 243918 5.591115389905 2013504.0 1864250.0 42368.0 145104.0 1852.0 1327.0 0.0 146075.0 1719146.0 92.6 2.1 7.2 0.1 0.1 0.0 7.3 85.4 50 50 50.00 38 100675200 26.4 22.9 23.1 27.6 0.0 37.4 24.7 smartseq 1452176 SRR4251144 SRP090061 SRS1698819 SRX2171145 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317363: 26Dn4_E02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317363 GSM2317363 26Dn4_E02_smart-seq 190014700 1900147 2016-09-30 15:56:31 129461780 190014700 1900147 2 1900147 index:0,count:1900147,average:50,stdev:0|index:1,count:1900147,average:50,stdev:0 GSM2317363_r1 2.75 3.49 0.14 166636353 208882043 157414413 198522212 125.35 126.11 1765965 1424988 252.510 2441.986 81 5527 71.54 75.86 1999047 1263443 1999047 1263443 72.54 72.96 1999047 1280977 1999047 1215203 22933218 13.76 1.95 0 5.28 0 0.10 0 0.13 0 0.00 0 6.83 0 1765965 0 100 0 98.71 0 1.40 0 0.01 0 1.22 0 0.01 0 273.62 0 0.24 0 37016 0 1900147 0 100389 0 1844 0 2491 0 0 0 129847 0 112 0 0 0 1365 0 202619 0 1414 0 205510 0 87.66 0 1665576 0 35717 210138 5.883416860319 1900147.0 1765965.0 37016.0 100389.0 1844.0 2491.0 0.0 129847.0 1665576.0 92.9 1.9 5.3 0.1 0.1 0.0 6.8 87.7 50 50 50.00 38 95007350 27.0 22.3 22.4 28.3 0.0 37.6 24.2 smartseq 1452177 SRR4252144 SRP090061 SRS1699821 SRX2172145 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318363: SK_3_G11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318363 GSM2318363 SK_3_G11_smart-seq 172258900 1722589 2016-09-30 15:56:31 119092072 172258900 1722589 2 1722589 index:0,count:1722589,average:50,stdev:0|index:1,count:1722589,average:50,stdev:0 GSM2318363_r1 6.43 2.9 0.05 148173564 198102374 138406004 186955526 133.7 135.08 1580798 1238817 242.393 2514.291 80 5503 77.93 83.62 1864797 1231961 1864797 1231961 78.79 79.97 1864797 1245542 1864797 1178146 14462830 9.76 2.00 0 6.24 0 0.07 0 0.07 0 0.00 0 8.09 0 1580798 0 100 0 98.49 0 1.34 0 0.01 0 1.21 0 0.01 0 269.62 0 0.26 0 34392 0 1722589 0 107568 0 1191 0 1170 0 0 0 139430 0 185 0 0 0 1454 0 203174 0 1065 0 205878 0 85.52 0 1473230 0 45825 208115 4.541516639389 1722589.0 1580798.0 34392.0 107568.0 1191.0 1170.0 0.0 139430.0 1473230.0 91.8 2.0 6.2 0.1 0.1 0.0 8.1 85.5 50 50 50.00 38 86129450 26.5 22.9 23.1 27.6 0.0 37.4 24.6 smartseq 1452208 SRR4252145 SRP090061 SRS1699824 SRX2172146 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318364: SK_3_G12_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;TRUE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318364 GSM2318364 SK_3_G12_smart-seq 222733500 2227335 2016-09-30 15:56:31 153255458 222733500 2227335 2 2227335 index:0,count:2227335,average:50,stdev:0|index:1,count:2227335,average:50,stdev:0 GSM2318364_r1 2.3 3.23 0.15 190563240 246185263 177616227 231983869 129.19 130.61 2040428 1638404 232.647 2239.803 83 7553 74.82 80.43 2415281 1526622 2415281 1526622 75.98 76.87 2415281 1550249 2415281 1459067 19340496 10.15 2.02 0 6.39 0 0.09 0 0.10 0 0.00 0 8.20 0 2040428 0 100 0 98.53 0 1.41 0 0.01 0 1.20 0 0.01 0 235.84 0 0.26 0 44895 0 2227335 0 142318 0 2036 0 2338 0 0 0 182533 0 237 0 0 0 1880 0 249466 0 1461 0 253044 0 85.22 0 1898110 0 41658 256424 6.155456334918 2227335.0 2040428.0 44895.0 142318.0 2036.0 2338.0 0.0 182533.0 1898110.0 91.6 2.0 6.4 0.1 0.1 0.0 8.2 85.2 50 50 50.00 38 111366750 26.6 22.7 22.8 27.9 0.0 37.5 24.6 smartseq 1452752 SRR4252156 SRP090061 SRS1699833 SRX2172157 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2318375: SK_3_H11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2318375 GSM2318375 SK_3_H11_smart-seq 119043500 1190435 2016-09-30 15:56:31 82124652 119043500 1190435 2 1190435 index:0,count:1190435,average:50,stdev:0|index:1,count:1190435,average:50,stdev:0 GSM2318375_r1 0.63 1.5 0.2 75517861 78812322 73322216 76890612 104.36 104.87 879742 851715 142.819 1050.472 57 7250 86.95 89.92 975230 764943 975230 764943 87.17 88.95 975230 766899 975230 756724 2795730 3.70 2.17 0 2.44 0 0.18 0 0.05 0 0.00 0 25.87 0 879742 0 100 0 97.08 0 1.49 0 0.01 0 1.12 0 0.01 0 214.28 0 0.21 0 25876 0 1190435 0 29047 0 2096 0 641 0 0 0 307956 0 1 0 0 0 84 0 22618 0 392 0 23095 0 71.46 0 850695 0 1834 22300 12.159214830971 1190435.0 879742.0 25876.0 29047.0 2096.0 641.0 0.0 307956.0 850695.0 73.9 2.2 2.4 0.2 0.1 0.0 25.9 71.5 50 50 50.00 38 59521750 25.4 21.1 21.1 32.3 0.0 36.6 18.9 smartseq 1454256 SRR4251185 SRP090061 SRS1698862 SRX2171186 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317404: 26Dn4_H10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317404 GSM2317404 26Dn4_H10_smart-seq 93184600 931846 2016-09-30 15:56:31 64271810 93184600 931846 2 931846 index:0,count:931846,average:50,stdev:0|index:1,count:931846,average:50,stdev:0 GSM2317404_r1 2.49 3.39 0.16 79548021 98328709 75662667 94019309 123.61 124.26 850078 686428 267.804 2451.144 68 2740 69.53 73.26 948183 591057 948183 591057 69.95 70.37 948183 594627 948183 567758 12620024 15.86 2.05 0 4.65 0 0.09 0 0.13 0 0.00 0 8.56 0 850078 0 100 0 98.63 0 1.40 0 0.01 0 1.24 0 0.01 0 186.37 0 0.25 0 19118 0 931846 0 43299 0 870 0 1172 0 0 0 79726 0 88 0 0 0 708 0 93417 0 637 0 94850 0 86.58 0 806779 0 29093 95945 3.297872340426 931846.0 850078.0 19118.0 43299.0 870.0 1172.0 0.0 79726.0 806779.0 91.2 2.1 4.6 0.1 0.1 0.0 8.6 86.6 50 50 50.00 38 46592300 26.8 22.5 22.5 28.1 0.0 37.4 24.2 smartseq 1454289 SRR4251186 SRP090061 SRS1698865 SRX2171187 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317405: 26Dn4_H11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317405 GSM2317405 26Dn4_H11_smart-seq 104432600 1044326 2016-09-30 15:56:31 71817004 104432600 1044326 2 1044326 index:0,count:1044326,average:50,stdev:0|index:1,count:1044326,average:50,stdev:0 GSM2317405_r1 3.49 3.2 0.12 89131901 111371080 84446716 105881708 124.95 125.38 944416 755606 277.875 2217.601 90 2727 71.24 75.35 1057019 672845 1057019 672845 72.11 72.47 1057019 681058 1057019 647187 13046730 14.64 2.20 0 4.92 0 0.08 0 0.09 0 0.00 0 9.40 0 944416 0 100 0 98.49 0 1.42 0 0.01 0 1.22 0 0.01 0 234.97 0 0.26 0 22962 0 1044326 0 51398 0 803 0 942 0 0 0 98165 0 70 0 0 0 760 0 101964 0 714 0 103508 0 85.51 0 893018 0 28683 104397 3.639682041627 1044326.0 944416.0 22962.0 51398.0 803.0 942.0 0.0 98165.0 893018.0 90.4 2.2 4.9 0.1 0.1 0.0 9.4 85.5 50 50 50.00 38 52216300 27.2 21.9 22.0 28.8 0.0 37.4 23.7 smartseq 1454320 SRR4251187 SRP090061 SRS1698864 SRX2171188 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317406: 54Dp4_A01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317406 GSM2317406 54Dp4_A01_smart-seq 110840300 1108403 2016-09-30 15:56:31 75034355 110840300 1108403 2 1108403 index:0,count:1108403,average:50,stdev:0|index:1,count:1108403,average:50,stdev:0 GSM2317406_r1 2.68 3.24 0.09 89500816 107618943 85188914 103419611 120.24 121.4 932213 771704 292.261 2913.645 136 2438 60.39 63.53 1073974 562938 1073974 562938 60.7 61.2 1073974 565811 1073974 542304 19477522 21.76 1.58 0 4.16 0 0.10 0 0.10 0 0.00 0 15.69 0 932213 0 100 0 98.86 0 1.39 0 0.01 0 1.23 0 0.01 0 234.72 0 0.22 0 17534 0 1108403 0 46065 0 1084 0 1150 0 0 0 173956 0 48 0 0 0 583 0 77003 0 876 0 78510 0 79.95 0 886148 0 26146 80530 3.080012238966 1108403.0 932213.0 17534.0 46065.0 1084.0 1150.0 0.0 173956.0 886148.0 84.1 1.6 4.2 0.1 0.1 0.0 15.7 79.9 50 50 50.00 38 55420150 27.3 21.9 22.2 28.7 0.0 37.8 24.1 smartseq 1461264 SRR4251260 SRP090061 SRS1698937 SRX2171261 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317479: 54Dp4_G11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317479 GSM2317479 54Dp4_G11_smart-seq 196007200 1960072 2016-09-30 15:56:31 127435843 196007200 1960072 2 1960072 index:0,count:1960072,average:50,stdev:0|index:1,count:1960072,average:50,stdev:0 GSM2317479_r1 3.04 2.63 0.16 169084957 198218777 161188402 190716251 117.23 118.32 1778838 1572784 255.942 1944.966 111 5789 51.17 53.75 2033847 910216 2033847 910216 51.5 51.66 2033847 916015 2033847 874689 47980516 28.38 1.87 0 4.36 0 0.10 0 0.15 0 0.00 0 9.00 0 1778838 0 100 0 98.74 0 1.40 0 0.01 0 1.21 0 0.01 0 252.01 0 0.18 0 36641 0 1960072 0 85547 0 2016 0 2900 0 0 0 176318 0 94 0 0 0 839 0 108408 0 1453 0 110794 0 86.39 0 1693291 0 23227 113111 4.869806690490 1960072.0 1778838.0 36641.0 85547.0 2016.0 2900.0 0.0 176318.0 1693291.0 90.8 1.9 4.4 0.1 0.1 0.0 9.0 86.4 50 50 50.00 38 98003600 28.4 20.5 20.7 30.3 0.0 38.1 24.6 smartseq 1461329 SRR4251262 SRP090061 SRS1698939 SRX2171263 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317481: 54Dp4_H02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317481 GSM2317481 54Dp4_H02_smart-seq 117094600 1170946 2016-09-30 15:56:31 78301783 117094600 1170946 2 1170946 index:0,count:1170946,average:50,stdev:0|index:1,count:1170946,average:50,stdev:0 GSM2317481_r1 2.13 2.81 0.16 89884798 100897786 85862484 97539940 112.25 113.6 931432 826998 299.665 1987.626 145 2534 42.04 44.06 1078250 391544 1078250 391544 42.27 42.53 1078250 393693 1078250 377945 30070833 33.45 2.06 0 3.66 0 0.11 0 0.15 0 0.00 0 20.19 0 931432 0 100 0 98.88 0 1.44 0 0.01 0 1.20 0 0.01 0 281.03 0 0.20 0 24084 0 1170946 0 42817 0 1315 0 1756 0 0 0 236443 0 43 0 0 0 410 0 43939 0 894 0 45286 0 75.89 0 888615 0 16290 45965 2.821669736034 1170946.0 931432.0 24084.0 42817.0 1315.0 1756.0 0.0 236443.0 888615.0 79.5 2.1 3.7 0.1 0.1 0.0 20.2 75.9 50 50 50.00 38 58547300 27.9 21.0 21.4 29.7 0.0 37.8 23.6 smartseq 1461360 SRR4251263 SRP090061 SRS1698943 SRX2171264 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317482: 54Dp4_H03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317482 GSM2317482 54Dp4_H03_smart-seq 102406800 1024068 2016-09-30 15:56:31 69478538 102406800 1024068 2 1024068 index:0,count:1024068,average:50,stdev:0|index:1,count:1024068,average:50,stdev:0 GSM2317482_r1 3.17 3.31 0.11 82398090 103174104 78134897 98889021 125.21 126.56 857135 686538 304.364 3054.301 134 2137 67.33 71.11 996187 577112 996187 577112 67.56 68.38 996187 579072 996187 554930 13613692 16.52 1.92 0 4.45 0 0.08 0 0.09 0 0.00 0 16.14 0 857135 0 100 0 98.68 0 1.38 0 0.01 0 1.22 0 0.01 0 230.42 0 0.22 0 19635 0 1024068 0 45583 0 770 0 909 0 0 0 165254 0 87 0 0 0 677 0 78623 0 703 0 80090 0 79.25 0 811552 0 26349 81872 3.107214695055 1024068.0 857135.0 19635.0 45583.0 770.0 909.0 0.0 165254.0 811552.0 83.7 1.9 4.5 0.1 0.1 0.0 16.1 79.2 50 50 50.00 38 51203400 27.4 21.7 22.1 28.8 0.0 37.8 24.0 smartseq 1461457 SRR4251266 SRP090061 SRS1698942 SRX2171267 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317485: 54Dp4_H06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317485 GSM2317485 54Dp4_H06_smart-seq 157150400 1571504 2016-09-30 15:56:31 104081783 157150400 1571504 2 1571504 index:0,count:1571504,average:50,stdev:0|index:1,count:1571504,average:50,stdev:0 GSM2317485_r1 4.68 3.23 0.25 126764272 149885615 120026183 143741597 118.24 119.76 1338358 1169674 262.939 2048.848 113 4222 54.25 57.4 1590966 726019 1590966 726019 54.65 55.26 1590966 731419 1590966 698910 31706859 25.01 1.94 0 4.68 0 0.08 0 0.12 0 0.00 0 14.63 0 1338358 0 100 0 98.70 0 1.34 0 0.01 0 1.20 0 0.01 0 209.53 0 0.19 0 30500 0 1571504 0 73618 0 1236 0 1946 0 0 0 229964 0 50 0 0 0 651 0 86616 0 993 0 88310 0 80.48 0 1264740 0 19977 89600 4.485157931621 1571504.0 1338358.0 30500.0 73618.0 1236.0 1946.0 0.0 229964.0 1264740.0 85.2 1.9 4.7 0.1 0.1 0.0 14.6 80.5 50 50 50.00 38 78575200 27.8 21.1 21.4 29.7 0.0 37.8 23.7 smartseq 1461490 SRR4251267 SRP090061 SRS1698945 SRX2171268 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317486: 54Dp4_H07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317486 GSM2317486 54Dp4_H07_smart-seq 151847200 1518472 2016-09-30 15:56:31 100904982 151847200 1518472 2 1518472 index:0,count:1518472,average:50,stdev:0|index:1,count:1518472,average:50,stdev:0 GSM2317486_r1 5.45 2.42 0.2 125455441 156820758 116051872 148157968 125.0 127.67 1309924 1103986 286.089 2417.921 134 3634 65.06 70.51 1681925 852300 1681925 852300 65.92 67.73 1681925 863528 1681925 818682 21432761 17.08 1.97 0 6.66 0 0.06 0 0.08 0 0.00 0 13.59 0 1309924 0 100 0 98.67 0 1.35 0 0.01 0 1.19 0 0.01 0 273.32 0 0.19 0 29846 0 1518472 0 101119 0 985 0 1236 0 0 0 206327 0 91 0 0 0 731 0 101570 0 895 0 103287 0 79.61 0 1208805 0 21589 105862 4.903515679281 1518472.0 1309924.0 29846.0 101119.0 985.0 1236.0 0.0 206327.0 1208805.0 86.3 2.0 6.7 0.1 0.1 0.0 13.6 79.6 50 50 50.00 38 75923600 27.5 21.4 21.7 29.4 0.0 37.8 23.8 smartseq 1461522 SRR4251268 SRP090061 SRS1698944 SRX2171269 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317487: 54Dp4_H08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317487 GSM2317487 54Dp4_H08_smart-seq 153825100 1538251 2016-09-30 15:56:31 102107831 153825100 1538251 2 1538251 index:0,count:1538251,average:50,stdev:0|index:1,count:1538251,average:50,stdev:0 GSM2317487_r1 3.22 2.75 0.12 132326255 160274749 125431233 153788712 121.12 122.61 1378902 1150088 279.897 2636.424 137 3922 61.43 64.89 1617926 847062 1617926 847062 61.71 62.29 1617926 850869 1617926 813026 29701294 22.45 1.65 0 4.79 0 0.06 0 0.10 0 0.00 0 10.19 0 1378902 0 100 0 98.77 0 1.34 0 0.01 0 1.19 0 0.01 0 240.77 0 0.19 0 25363 0 1538251 0 73608 0 929 0 1596 0 0 0 156824 0 90 0 0 0 983 0 117760 0 935 0 119768 0 84.86 0 1305294 0 28286 121828 4.307006999929 1538251.0 1378902.0 25363.0 73608.0 929.0 1596.0 0.0 156824.0 1305294.0 89.6 1.6 4.8 0.1 0.1 0.0 10.2 84.9 50 50 50.00 38 76912550 27.5 21.6 21.8 29.1 0.0 37.9 24.7 smartseq 1461553 SRR4251269 SRP090061 SRS1698947 SRX2171270 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317488: 54Dp4_H09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317488 GSM2317488 54Dp4_H09_smart-seq 143470600 1434706 2016-09-30 15:56:31 94466546 143470600 1434706 2 1434706 index:0,count:1434706,average:50,stdev:0|index:1,count:1434706,average:50,stdev:0 GSM2317488_r1 5.65 2.65 0.17 123084282 152297515 116451296 145598511 123.73 125.03 1284400 1069445 281.635 2769.855 130 3544 64.01 67.76 1508177 822132 1508177 822132 64.7 65.23 1508177 831046 1508177 791446 23873311 19.40 1.76 0 4.95 0 0.08 0 0.11 0 0.00 0 10.29 0 1284400 0 100 0 98.75 0 1.35 0 0.01 0 1.20 0 0.01 0 286.94 0 0.19 0 25183 0 1434706 0 71015 0 1121 0 1570 0 0 0 147615 0 78 0 0 0 867 0 107805 0 1062 0 109812 0 84.57 0 1213385 0 26392 112396 4.258714762049 1434706.0 1284400.0 25183.0 71015.0 1121.0 1570.0 0.0 147615.0 1213385.0 89.5 1.8 4.9 0.1 0.1 0.0 10.3 84.6 50 50 50.00 38 71735300 27.4 21.8 21.9 28.8 0.0 38.0 24.7 smartseq 1461777 SRR4251270 SRP090061 SRS1698946 SRX2171271 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317489: 54Dp4_H10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317489 GSM2317489 54Dp4_H10_smart-seq 171371900 1713719 2016-09-30 15:56:31 112964259 171371900 1713719 2 1713719 index:0,count:1713719,average:50,stdev:0|index:1,count:1713719,average:50,stdev:0 GSM2317489_r1 1.86 2.49 0.16 137292221 158693129 130373737 152376825 115.59 116.88 1456863 1315080 247.979 1735.240 100 5180 50.36 53.12 1703248 733731 1703248 733731 50.83 51.22 1703248 740455 1703248 707506 38662562 28.16 1.93 0 4.41 0 0.11 0 0.18 0 0.00 0 14.70 0 1456863 0 100 0 98.72 0 1.40 0 0.01 0 1.19 0 0.01 0 228.50 0 0.18 0 33006 0 1713719 0 75656 0 1864 0 3062 0 0 0 251930 0 93 0 0 0 636 0 75634 0 1027 0 77390 0 80.60 0 1381207 0 15189 78604 5.175060899335 1713719.0 1456863.0 33006.0 75656.0 1864.0 3062.0 0.0 251930.0 1381207.0 85.0 1.9 4.4 0.1 0.2 0.0 14.7 80.6 50 50 50.00 38 85685950 28.0 20.9 21.1 30.1 0.0 38.0 23.9 smartseq 1461809 SRR4251271 SRP090061 SRS1698950 SRX2171272 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317490: 54Dp4_H11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317490 GSM2317490 54Dp4_H11_smart-seq 260045500 2600455 2016-09-30 15:56:31 168715579 260045500 2600455 2 2600455 index:0,count:2600455,average:50,stdev:0|index:1,count:2600455,average:50,stdev:0 GSM2317490_r1 6.63 1.9 0.06 210243935 264073904 190356486 247672626 125.6 130.11 2282267 2035906 202.362 1564.803 78 10229 64.15 71.12 3232520 1464048 3232520 1464048 65.03 68.15 3232520 1484161 3232520 1402906 34544821 16.43 2.04 0 8.60 0 0.10 0 0.10 0 0.00 0 12.04 0 2282267 0 100 0 98.38 0 1.34 0 0.01 0 1.19 0 0.01 0 390.07 0 0.18 0 52925 0 2600455 0 223739 0 2535 0 2478 0 0 0 313175 0 137 0 0 0 1335 0 169460 0 1300 0 172232 0 79.16 0 2058528 0 21058 174559 8.289438693133 2600455.0 2282267.0 52925.0 223739.0 2535.0 2478.0 0.0 313175.0 2058528.0 87.8 2.0 8.6 0.1 0.1 0.0 12.0 79.2 50 50 50.00 38 130022750 27.2 21.6 21.8 29.4 0.0 38.0 23.9 smartseq 1461841 SRR4251272 SRP090061 SRS1698948 SRX2171273 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317491: 54Dn4_A01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317491 GSM2317491 54Dn4_A01_smart-seq 146568200 1465682 2016-09-30 15:56:31 99816423 146568200 1465682 2 1465682 index:0,count:1465682,average:50,stdev:0|index:1,count:1465682,average:50,stdev:0 GSM2317491_r1 2.38 2.54 0.13 125258716 153083801 120064470 147620842 122.21 122.95 1314852 1105831 271.056 2415.172 105 3964 66.02 68.97 1468814 868097 1468814 868097 65.87 66.41 1468814 866031 1468814 835864 23959823 19.13 1.64 0 3.84 0 0.08 0 0.11 0 0.00 0 10.11 0 1314852 0 100 0 98.79 0 1.39 0 0.01 0 1.21 0 0.01 0 251.26 0 0.22 0 24039 0 1465682 0 56227 0 1170 0 1542 0 0 0 148118 0 46 0 0 0 899 0 109011 0 1140 0 111096 0 85.87 0 1258625 0 25425 113223 4.453215339233 1465682.0 1314852.0 24039.0 56227.0 1170.0 1542.0 0.0 148118.0 1258625.0 89.7 1.6 3.8 0.1 0.1 0.0 10.1 85.9 50 50 50.00 38 73284100 27.7 21.3 21.5 29.6 0.0 37.6 23.5 smartseq 1461872 SRR4251273 SRP090061 SRS1698949 SRX2171274 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317492: 54Dn4_A02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317492 GSM2317492 54Dn4_A02_smart-seq 188751000 1887510 2016-09-30 15:56:31 128658806 188751000 1887510 2 1887510 index:0,count:1887510,average:50,stdev:0|index:1,count:1887510,average:50,stdev:0 GSM2317492_r1 2.29 3.07 0.08 166805727 205997055 158687733 197591332 123.5 124.52 1725973 1380464 291.456 2611.282 137 4470 66.29 69.74 1956525 1144156 1956525 1144156 66.59 67.05 1956525 1149391 1956525 1100023 29098343 17.44 1.67 0 4.52 0 0.09 0 0.09 0 0.00 0 8.37 0 1725973 0 100 0 98.86 0 1.38 0 0.01 0 1.21 0 0.01 0 234.31 0 0.23 0 31554 0 1887510 0 85392 0 1779 0 1686 0 0 0 158072 0 129 0 0 0 1172 0 175616 0 1453 0 178370 0 86.92 0 1640581 0 36025 182654 5.070201249133 1887510.0 1725973.0 31554.0 85392.0 1779.0 1686.0 0.0 158072.0 1640581.0 91.4 1.7 4.5 0.1 0.1 0.0 8.4 86.9 50 50 50.00 38 94375500 27.4 21.7 22.0 28.8 0.0 37.7 24.4 smartseq 1466896 SRR4251310 SRP090061 SRS1698988 SRX2171311 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317529: 54Dn4_D06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317529 GSM2317529 54Dn4_D06_smart-seq 152815300 1528153 2016-09-30 15:56:31 104806783 152815300 1528153 2 1528153 index:0,count:1528153,average:50,stdev:0|index:1,count:1528153,average:50,stdev:0 GSM2317529_r1 1.71 3.23 0.09 136814822 171990024 130324500 165145537 125.71 126.72 1431718 1127450 269.643 2849.451 109 4009 71.48 75.12 1613896 1023333 1613896 1023333 71.51 72.14 1613896 1023804 1613896 982858 20004304 14.62 1.71 0 4.54 0 0.06 0 0.11 0 0.00 0 6.13 0 1431718 0 100 0 98.76 0 1.40 0 0.01 0 1.22 0 0.01 0 289.54 0 0.23 0 26124 0 1528153 0 69375 0 992 0 1706 0 0 0 93737 0 98 0 0 0 1347 0 169328 0 1149 0 171922 0 89.15 0 1362343 0 39107 174613 4.465006264863 1528153.0 1431718.0 26124.0 69375.0 992.0 1706.0 0.0 93737.0 1362343.0 93.7 1.7 4.5 0.1 0.1 0.0 6.1 89.1 50 50 50.00 38 76407650 27.3 21.9 22.1 28.6 0.0 37.7 24.7 smartseq 1466992 SRR4251313 SRP090061 SRS1698990 SRX2171314 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317532: 54Dn4_D09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317532 GSM2317532 54Dn4_D09_smart-seq 145247500 1452475 2016-09-30 15:56:31 98923468 145247500 1452475 2 1452475 index:0,count:1452475,average:50,stdev:0|index:1,count:1452475,average:50,stdev:0 GSM2317532_r1 4.57 3.59 0.07 130480959 165772470 124846320 159634243 127.05 127.86 1363661 1088718 274.107 2845.747 115 3819 69.18 72.38 1523519 943330 1523519 943330 69.2 69.76 1523519 943706 1523519 909178 21718169 16.64 1.69 0 4.16 0 0.09 0 0.09 0 0.00 0 5.93 0 1363661 0 100 0 98.79 0 1.36 0 0.01 0 1.22 0 0.01 0 249.00 0 0.23 0 24542 0 1452475 0 60444 0 1318 0 1329 0 0 0 86167 0 99 0 0 0 1137 0 147608 0 1229 0 150073 0 89.72 0 1303217 0 37925 152911 4.031931443639 1452475.0 1363661.0 24542.0 60444.0 1318.0 1329.0 0.0 86167.0 1303217.0 93.9 1.7 4.2 0.1 0.1 0.0 5.9 89.7 50 50 50.00 38 72623750 27.4 21.9 22.1 28.6 0.0 37.7 24.8 smartseq 1467024 SRR4251314 SRP090061 SRS1698991 SRX2171315 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317533: 54Dn4_D10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317533 GSM2317533 54Dn4_D10_smart-seq 186237300 1862373 2016-09-30 15:56:31 127143872 186237300 1862373 2 1862373 index:0,count:1862373,average:50,stdev:0|index:1,count:1862373,average:50,stdev:0 GSM2317533_r1 3.93 2.54 0.12 150531317 186345744 143044208 178472645 123.79 124.77 1584399 1358417 247.610 2226.108 100 5407 65.76 69.36 1842687 1041863 1842687 1041863 65.94 66.81 1842687 1044777 1842687 1003461 25036844 16.63 1.43 0 4.42 0 0.09 0 0.11 0 0.00 0 14.73 0 1584399 0 100 0 98.86 0 1.37 0 0.01 0 1.20 0 0.01 0 319.26 0 0.22 0 26712 0 1862373 0 82332 0 1660 0 2068 0 0 0 274246 0 140 0 0 0 1173 0 129065 0 1262 0 131640 0 80.65 0 1502067 0 23170 135488 5.847561501942 1862373.0 1584399.0 26712.0 82332.0 1660.0 2068.0 0.0 274246.0 1502067.0 85.1 1.4 4.4 0.1 0.1 0.0 14.7 80.7 50 50 50.00 38 93118650 27.3 21.7 21.8 29.2 0.0 37.7 24.0 smartseq 1467056 SRR4250315 SRP090061 SRS1697992 SRX2170316 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316534: 26Dp1_A04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316534 GSM2316534 26Dp1_A04_smart-seq 79638800 796388 2016-09-30 15:56:31 35563042 79638800 796388 2 796388 index:0,count:796388,average:50,stdev:0|index:1,count:796388,average:50,stdev:0 GSM2316534_r1 2.85 3.1 0.13 68482251 87127619 64415469 83038137 127.23 128.91 732942 608432 234.997 2436.214 78 2698 71.39 76.06 876033 523259 876033 523259 71.23 72.49 876033 522105 876033 498678 9158214 13.37 1.86 0 5.65 0 0.12 0 0.13 0 0.00 0 7.71 0 732942 0 100 0 98.62 0 1.29 0 0.01 0 1.17 0 0.01 0 168.65 0 0.23 0 14826 0 796388 0 44990 0 976 0 1037 0 0 0 61433 0 56 0 0 0 596 0 76200 0 474 0 77326 0 86.38 0 687952 0 25309 78759 3.111896953653 796388.0 732942.0 14826.0 44990.0 976.0 1037.0 0.0 61433.0 687952.0 92.0 1.9 5.6 0.1 0.1 0.0 7.7 86.4 50 50 50.00 28 39819400 26.7 22.3 22.4 28.5 0.0 36.6 25.9 smartseq 1467057 SRR4251315 SRP090061 SRS1698992 SRX2171316 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317534: 54Dn4_D11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317534 GSM2317534 54Dn4_D11_smart-seq 201894800 2018948 2016-09-30 15:56:31 138098892 201894800 2018948 2 2018948 index:0,count:2018948,average:50,stdev:0|index:1,count:2018948,average:50,stdev:0 GSM2317534_r1 2.7 2.71 0.11 180417579 227355426 171435484 217567116 126.02 126.91 1881871 1484593 273.127 2653.297 113 5124 71.72 75.56 2136023 1349668 2136023 1349668 71.94 72.67 2136023 1353794 2136023 1297966 26200348 14.52 1.67 0 4.74 0 0.06 0 0.08 0 0.00 0 6.65 0 1881871 0 100 0 98.77 0 1.41 0 0.01 0 1.24 0 0.01 0 363.41 0 0.23 0 33670 0 2018948 0 95751 0 1231 0 1597 0 0 0 134249 0 212 0 0 0 1498 0 220834 0 1544 0 224088 0 88.47 0 1786120 0 42115 227112 5.392662946694 2018948.0 1881871.0 33670.0 95751.0 1231.0 1597.0 0.0 134249.0 1786120.0 93.2 1.7 4.7 0.1 0.1 0.0 6.6 88.5 50 50 50.00 38 100947400 27.4 21.9 22.1 28.7 0.0 37.7 24.6 smartseq 1467088 SRR4251316 SRP090061 SRS1698994 SRX2171317 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317535: 54Dn4_E01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317535 GSM2317535 54Dn4_E01_smart-seq 247792600 2477926 2016-09-30 15:56:31 165860188 247792600 2477926 2 2477926 index:0,count:2477926,average:50,stdev:0|index:1,count:2477926,average:50,stdev:0 GSM2317535_r1 1.34 2.92 0.18 221940697 266466666 213262255 257550866 120.06 120.77 2314442 1946245 265.371 2390.031 137 6779 61.11 63.66 2559168 1414367 2559168 1414367 60.95 61.21 2559168 1410693 2559168 1359923 48889269 22.03 1.63 0 3.74 0 0.07 0 0.18 0 0.00 0 6.35 0 2314442 0 100 0 98.91 0 1.43 0 0.01 0 1.20 0 0.01 0 318.59 0 0.20 0 40365 0 2477926 0 92580 0 1756 0 4346 0 0 0 157382 0 162 0 0 0 1472 0 197811 0 2058 0 201503 0 89.67 0 2221862 0 31822 207590 6.523474325938 2477926.0 2314442.0 40365.0 92580.0 1756.0 4346.0 0.0 157382.0 2221862.0 93.4 1.6 3.7 0.1 0.2 0.0 6.4 89.7 50 50 50.00 38 123896300 27.7 21.5 21.7 29.2 0.0 37.8 24.8 smartseq 1467120 SRR4251317 SRP090061 SRS1698993 SRX2171318 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317536: 54Dn4_E02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317536 GSM2317536 54Dn4_E02_smart-seq 156133900 1561339 2016-09-30 15:56:31 106087105 156133900 1561339 2 1561339 index:0,count:1561339,average:50,stdev:0|index:1,count:1561339,average:50,stdev:0 GSM2317536_r1 3.38 3.2 0.09 139501869 176533351 132683018 169741522 126.55 127.93 1455660 1136758 285.391 3099.133 103 3885 70.87 74.61 1679064 1031605 1679064 1031605 70.75 71.68 1679064 1029905 1679064 991085 21630865 15.51 1.78 0 4.67 0 0.08 0 0.12 0 0.00 0 6.57 0 1455660 0 100 0 98.74 0 1.39 0 0.01 0 1.21 0 0.01 0 216.19 0 0.23 0 27830 0 1561339 0 72918 0 1299 0 1871 0 0 0 102509 0 124 0 0 0 1133 0 165880 0 1113 0 168250 0 88.56 0 1382742 0 41850 171572 4.099689366786 1561339.0 1455660.0 27830.0 72918.0 1299.0 1871.0 0.0 102509.0 1382742.0 93.2 1.8 4.7 0.1 0.1 0.0 6.6 88.6 50 50 50.00 38 78066950 27.1 22.1 22.3 28.5 0.0 37.6 24.3 smartseq 1467152 SRR4251318 SRP090061 SRS1698999 SRX2171319 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317537: 54Dn4_E03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317537 GSM2317537 54Dn4_E03_smart-seq 228580600 2285806 2016-09-30 15:56:31 154327723 228580600 2285806 2 2285806 index:0,count:2285806,average:50,stdev:0|index:1,count:2285806,average:50,stdev:0 GSM2317537_r1 0.45 2.91 0.17 199283323 240452083 190340824 231432998 120.66 121.59 2088707 1762747 255.575 2227.622 134 6673 63.62 66.67 2342410 1328887 2342410 1328887 63.39 63.82 2342410 1324029 2342410 1272016 40103141 20.12 1.66 0 4.18 0 0.10 0 0.12 0 0.00 0 8.41 0 2088707 0 100 0 98.82 0 1.40 0 0.01 0 1.21 0 0.01 0 265.45 0 0.20 0 38013 0 2285806 0 95499 0 2172 0 2669 0 0 0 192258 0 158 0 0 0 1461 0 180759 0 1708 0 184086 0 87.20 0 1993208 0 28320 190061 6.711193502825 2285806.0 2088707.0 38013.0 95499.0 2172.0 2669.0 0.0 192258.0 1993208.0 91.4 1.7 4.2 0.1 0.1 0.0 8.4 87.2 50 50 50.00 38 114290300 27.5 21.5 21.6 29.3 0.0 37.8 24.2 smartseq 1467184 SRR4250319 SRP090061 SRS1697996 SRX2170320 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316538: 26Dp1_A08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316538 GSM2316538 26Dp1_A08_smart-seq 114588100 1145881 2016-09-30 15:56:31 50636736 114588100 1145881 2 1145881 index:0,count:1145881,average:50,stdev:0|index:1,count:1145881,average:50,stdev:0 GSM2316538_r1 4.21 2.99 0.11 96435930 124161501 89101236 116822933 128.75 131.11 1043512 890863 207.341 2013.766 78 4640 71.82 77.96 1299204 749470 1299204 749470 72.67 74.18 1299204 758366 1299204 713146 11370540 11.79 2.00 0 7.17 0 0.10 0 0.11 0 0.00 0 8.73 0 1043512 0 100 0 98.50 0 1.28 0 0.01 0 1.18 0 0.01 0 294.66 0 0.23 0 22885 0 1145881 0 82132 0 1121 0 1212 0 0 0 100036 0 104 0 0 0 796 0 111855 0 749 0 113504 0 83.90 0 961380 0 26644 115448 4.332983035580 1145881.0 1043512.0 22885.0 82132.0 1121.0 1212.0 0.0 100036.0 961380.0 91.1 2.0 7.2 0.1 0.1 0.0 8.7 83.9 50 50 50.00 28 57294050 26.3 22.6 22.7 28.3 0.0 36.7 25.6 smartseq 1467185 SRR4251319 SRP090061 SRS1698995 SRX2171320 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317538: 54Dn4_E04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317538 GSM2317538 54Dn4_E04_smart-seq 28470100 284701 2016-09-30 15:56:31 19650671 28470100 284701 2 284701 index:0,count:284701,average:50,stdev:0|index:1,count:284701,average:50,stdev:0 GSM2317538_r1 3.79 2.78 0.08 25413498 31802400 24136441 30430535 125.14 126.08 264428 209540 313.657 2900.942 110 682 66.54 70.15 299393 175942 299393 175942 67.24 67.66 299393 177809 299393 169687 4511565 17.75 1.91 0 4.79 0 0.08 0 0.09 0 0.00 0 6.95 0 264428 0 100 0 98.78 0 1.44 0 0.01 0 1.21 0 0.01 0 170.82 0 0.23 0 5432 0 284701 0 13635 0 225 0 252 0 0 0 19796 0 25 0 0 0 199 0 25795 0 199 0 26218 0 88.09 0 250793 0 14527 25830 1.778068424313 284701.0 264428.0 5432.0 13635.0 225.0 252.0 0.0 19796.0 250793.0 92.9 1.9 4.8 0.1 0.1 0.0 7.0 88.1 50 50 50.00 38 14235050 27.9 21.2 21.4 29.5 0.0 37.4 23.7 smartseq 1467409 SRR4250320 SRP090061 SRS1697997 SRX2170321 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316539: 26Dp1_A09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316539 GSM2316539 26Dp1_A09_smart-seq 76120500 761205 2016-09-30 15:56:31 33034028 76120500 761205 2 761205 index:0,count:761205,average:50,stdev:0|index:1,count:761205,average:50,stdev:0 GSM2316539_r1 3.78 3.54 0.1 63712929 83118462 58923059 77905127 130.46 132.22 694503 591369 210.057 1904.262 78 3213 73.11 79.32 848084 507728 848084 507728 74.38 75.57 848084 516595 848084 483728 7026498 11.03 2.21 0 7.14 0 0.11 0 0.13 0 0.00 0 8.52 0 694503 0 100 0 98.43 0 1.29 0 0.01 0 1.17 0 0.01 0 195.74 0 0.23 0 16816 0 761205 0 54367 0 853 0 970 0 0 0 64879 0 62 0 0 0 522 0 74659 0 482 0 75725 0 84.10 0 640136 0 23477 76004 3.237381266772 761205.0 694503.0 16816.0 54367.0 853.0 970.0 0.0 64879.0 640136.0 91.2 2.2 7.1 0.1 0.1 0.0 8.5 84.1 50 50 50.00 28 38060250 26.3 22.8 22.8 28.1 0.0 36.7 26.2 smartseq 1467410 SRR4251320 SRP090061 SRS1698998 SRX2171321 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317539: 54Dn4_E05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317539 GSM2317539 54Dn4_E05_smart-seq 194126100 1941261 2016-09-30 15:56:31 130216417 194126100 1941261 2 1941261 index:0,count:1941261,average:50,stdev:0|index:1,count:1941261,average:50,stdev:0 GSM2317539_r1 0.61 2.94 0.32 174934921 204252646 168744923 198025028 116.76 117.35 1817649 1533696 286.132 2636.883 134 5014 55.77 57.87 1987688 1013769 1987688 1013769 55.51 55.7 1987688 1009066 1987688 975885 46228668 26.43 1.62 0 3.39 0 0.08 0 0.17 0 0.00 0 6.12 0 1817649 0 100 0 98.96 0 1.47 0 0.01 0 1.21 0 0.01 0 279.54 0 0.20 0 31481 0 1941261 0 65738 0 1614 0 3267 0 0 0 118731 0 138 0 0 0 1102 0 141020 0 1566 0 143826 0 90.25 0 1751911 0 31050 148419 4.780000000000 1941261.0 1817649.0 31481.0 65738.0 1614.0 3267.0 0.0 118731.0 1751911.0 93.6 1.6 3.4 0.1 0.2 0.0 6.1 90.2 50 50 50.00 38 97063050 28.0 21.1 21.2 29.6 0.0 37.7 24.4 smartseq 1467440 SRR4250321 SRP090061 SRS1697999 SRX2170322 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316540: 26Dp1_A10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316540 GSM2316540 26Dp1_A10_smart-seq 62208000 622080 2016-09-30 15:56:31 28142635 62208000 622080 2 622080 index:0,count:622080,average:50,stdev:0|index:1,count:622080,average:50,stdev:0 GSM2316540_r1 2.21 3.24 0.18 52754713 68437394 48875707 64466813 129.73 131.9 566877 470228 236.992 2342.128 78 2202 74.95 81.15 699740 424870 699740 424870 75.87 77.44 699740 430114 699740 405420 5553886 10.53 2.03 0 6.97 0 0.09 0 0.09 0 0.00 0 8.69 0 566877 0 100 0 98.48 0 1.28 0 0.01 0 1.19 0 0.01 0 172.27 0 0.24 0 12656 0 622080 0 43330 0 587 0 581 0 0 0 54035 0 54 0 0 0 498 0 62298 0 504 0 63354 0 84.16 0 523547 0 23011 63760 2.770848724523 622080.0 566877.0 12656.0 43330.0 587.0 581.0 0.0 54035.0 523547.0 91.1 2.0 7.0 0.1 0.1 0.0 8.7 84.2 50 50 50.00 28 31104000 26.6 22.3 22.4 28.6 0.0 36.6 25.6 smartseq 1467441 SRR4251321 SRP090061 SRS1698997 SRX2171322 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317540: 54Dn4_E06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317540 GSM2317540 54Dn4_E06_smart-seq 178260700 1782607 2016-09-30 15:56:31 120656115 178260700 1782607 2 1782607 index:0,count:1782607,average:50,stdev:0|index:1,count:1782607,average:50,stdev:0 GSM2317540_r1 5.85 2.62 0.07 158551372 207678181 149122287 197104028 130.98 132.18 1654732 1283489 282.658 2550.537 134 4363 75.61 80.51 1918147 1251131 1918147 1251131 75.98 76.99 1918147 1257255 1918147 1196393 17932252 11.31 1.96 0 5.65 0 0.06 0 0.07 0 0.00 0 7.04 0 1654732 0 100 0 98.65 0 1.35 0 0.01 0 1.20 0 0.01 0 279.02 0 0.22 0 34896 0 1782607 0 100760 0 1041 0 1274 0 0 0 125560 0 177 0 0 0 1291 0 201245 0 1276 0 203989 0 87.17 0 1553972 0 37498 207836 5.542588938077 1782607.0 1654732.0 34896.0 100760.0 1041.0 1274.0 0.0 125560.0 1553972.0 92.8 2.0 5.7 0.1 0.1 0.0 7.0 87.2 50 50 50.00 38 89130350 26.8 22.4 22.6 28.2 0.0 37.6 24.1 smartseq 1467472 SRR4250322 SRP090061 SRS1697998 SRX2170323 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316541: 26Dp1_A11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316541 GSM2316541 26Dp1_A11_smart-seq 95459500 954595 2016-09-30 15:56:31 42020717 95459500 954595 2 954595 index:0,count:954595,average:50,stdev:0|index:1,count:954595,average:50,stdev:0 GSM2316541_r1 2.26 3.31 0.15 78319053 100643671 72240289 94625226 128.5 130.99 852553 725034 207.027 1889.330 80 3987 69.96 76.09 1069865 596445 1069865 596445 71.18 72.58 1069865 606817 1069865 568955 10243659 13.08 2.18 0 7.20 0 0.15 0 0.14 0 0.00 0 10.40 0 852553 0 100 0 98.45 0 1.28 0 0.01 0 1.16 0 0.01 0 214.78 0 0.24 0 20831 0 954595 0 68695 0 1415 0 1364 0 0 0 99263 0 39 0 0 0 881 0 94473 0 642 0 96035 0 82.11 0 783858 0 24134 96615 4.003273390238 954595.0 852553.0 20831.0 68695.0 1415.0 1364.0 0.0 99263.0 783858.0 89.3 2.2 7.2 0.1 0.1 0.0 10.4 82.1 50 50 50.00 28 47729750 26.4 22.3 22.3 29.0 0.0 36.6 24.9 smartseq 1467473 SRR4251322 SRP090061 SRS1698996 SRX2171323 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317541: 54Dn4_E07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317541 GSM2317541 54Dn4_E07_smart-seq 163988600 1639886 2016-09-30 15:56:31 111133909 163988600 1639886 2 1639886 index:0,count:1639886,average:50,stdev:0|index:1,count:1639886,average:50,stdev:0 GSM2317541_r1 2.81 2.97 0.13 147183789 186306031 140048029 178159002 126.58 127.21 1530950 1245963 297.290 2660.532 134 3861 67.02 70.52 1717459 1026091 1717459 1026091 67.11 67.44 1717459 1027383 1717459 981242 26099917 17.73 1.76 0 4.63 0 0.06 0 0.08 0 0.00 0 6.50 0 1530950 0 100 0 98.83 0 1.40 0 0.01 0 1.23 0 0.01 0 203.57 0 0.21 0 28906 0 1639886 0 75961 0 979 0 1287 0 0 0 106670 0 110 0 0 0 1109 0 139682 0 1331 0 142232 0 88.73 0 1454989 0 33162 146877 4.429075447802 1639886.0 1530950.0 28906.0 75961.0 979.0 1287.0 0.0 106670.0 1454989.0 93.4 1.8 4.6 0.1 0.1 0.0 6.5 88.7 50 50 50.00 38 81994300 27.4 21.8 21.9 28.9 0.0 37.6 24.2 smartseq 1467505 SRR4250323 SRP090061 SRS1698000 SRX2170324 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316542: 26Dp1_B01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316542 GSM2316542 26Dp1_B01_smart-seq 120307700 1203077 2016-09-30 15:56:31 52660530 120307700 1203077 2 1203077 index:0,count:1203077,average:50,stdev:0|index:1,count:1203077,average:50,stdev:0 GSM2316542_r1 3.85 3.46 0.09 101473645 130545271 93428169 122509805 128.65 131.13 1095881 928104 215.031 1996.294 78 4890 71.21 77.57 1384087 780341 1384087 780341 72.62 74.18 1384087 795871 1384087 746197 12575742 12.39 2.01 0 7.47 0 0.09 0 0.11 0 0.00 0 8.71 0 1095881 0 100 0 98.54 0 1.26 0 0.01 0 1.18 0 0.01 0 254.77 0 0.23 0 24223 0 1203077 0 89930 0 1138 0 1279 0 0 0 104779 0 79 0 0 0 921 0 117798 0 710 0 119508 0 83.61 0 1005951 0 28692 120612 4.203680468423 1203077.0 1095881.0 24223.0 89930.0 1138.0 1279.0 0.0 104779.0 1005951.0 91.1 2.0 7.5 0.1 0.1 0.0 8.7 83.6 50 50 50.00 28 60153850 26.4 22.5 22.5 28.5 0.0 36.7 25.8 smartseq 1467506 SRR4251323 SRP090061 SRS1699000 SRX2171324 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317542: 54Dn4_E08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317542 GSM2317542 54Dn4_E08_smart-seq 142960300 1429603 2016-09-30 15:56:31 96984905 142960300 1429603 2 1429603 index:0,count:1429603,average:50,stdev:0|index:1,count:1429603,average:50,stdev:0 GSM2317542_r1 3.05 3.1 0.11 128643647 163187868 122686641 156771622 126.85 127.78 1339010 1039694 287.375 3236.237 134 3499 72.48 76.09 1510165 970574 1510165 970574 72.35 73.08 1510165 968753 1510165 932109 19323939 15.02 1.78 0 4.44 0 0.05 0 0.08 0 0.00 0 6.20 0 1339010 0 100 0 98.72 0 1.37 0 0.01 0 1.22 0 0.01 0 285.92 0 0.22 0 25473 0 1429603 0 63475 0 737 0 1151 0 0 0 88705 0 106 0 0 0 1168 0 154177 0 1026 0 156477 0 89.22 0 1275535 0 37010 158772 4.289975682248 1429603.0 1339010.0 25473.0 63475.0 737.0 1151.0 0.0 88705.0 1275535.0 93.7 1.8 4.4 0.1 0.1 0.0 6.2 89.2 50 50 50.00 38 71480150 27.2 22.0 22.2 28.5 0.0 37.6 24.3 smartseq 1467537 SRR4250324 SRP090061 SRS1698001 SRX2170325 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316543: 26Dp1_B02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316543 GSM2316543 26Dp1_B02_smart-seq 87460900 874609 2016-09-30 15:56:31 38247569 87460900 874609 2 874609 index:0,count:874609,average:50,stdev:0|index:1,count:874609,average:50,stdev:0 GSM2316543_r1 4.13 3.19 0.05 73726469 98175046 66711523 91026808 133.16 136.45 795808 657977 213.675 1862.312 88 3357 80.81 89.61 1041795 643105 1041795 643105 82.88 85.48 1041795 659575 1041795 613441 3957536 5.37 2.24 0 8.94 0 0.09 0 0.06 0 0.00 0 8.86 0 795808 0 100 0 98.40 0 1.25 0 0.01 0 1.17 0 0.01 0 242.20 0 0.23 0 19617 0 874609 0 78161 0 777 0 564 0 0 0 77460 0 64 0 0 0 744 0 100252 0 548 0 101608 0 82.05 0 717647 0 24856 102385 4.119126166720 874609.0 795808.0 19617.0 78161.0 777.0 564.0 0.0 77460.0 717647.0 91.0 2.2 8.9 0.1 0.1 0.0 8.9 82.1 50 50 50.00 28 43730450 25.8 23.1 23.1 27.9 0.0 36.7 25.7 smartseq 1467538 SRR4251324 SRP090061 SRS1699001 SRX2171325 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317543: 54Dn4_E09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317543 GSM2317543 54Dn4_E09_smart-seq 179192700 1791927 2016-09-30 15:56:31 119445401 179192700 1791927 2 1791927 index:0,count:1791927,average:50,stdev:0|index:1,count:1791927,average:50,stdev:0 GSM2317543_r1 1.69 3.04 0.14 159703473 197411824 150673619 187972394 123.61 124.75 1670983 1334446 270.464 2383.801 134 4732 67.97 72.13 1925920 1135704 1925920 1135704 68.47 69.09 1925920 1144121 1925920 1087749 26761273 16.76 1.91 0 5.38 0 0.09 0 0.09 0 0.00 0 6.57 0 1670983 0 100 0 98.70 0 1.43 0 0.01 0 1.21 0 0.01 0 268.79 0 0.20 0 34306 0 1791927 0 96481 0 1562 0 1666 0 0 0 117716 0 154 0 0 0 1496 0 184005 0 1361 0 187016 0 87.87 0 1574502 0 36611 192584 5.260276965939 1791927.0 1670983.0 34306.0 96481.0 1562.0 1666.0 0.0 117716.0 1574502.0 93.3 1.9 5.4 0.1 0.1 0.0 6.6 87.9 50 50 50.00 38 89596350 27.2 21.9 22.1 28.8 0.0 37.8 24.4 smartseq 1467568 SRR4251325 SRP090061 SRS1699003 SRX2171326 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317544: 54Dn4_E10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317544 GSM2317544 54Dn4_E10_smart-seq 218501500 2185015 2016-09-30 15:56:31 146484793 218501500 2185015 2 2185015 index:0,count:2185015,average:50,stdev:0|index:1,count:2185015,average:50,stdev:0 GSM2317544_r1 4.14 3.53 0.16 190961712 235156831 181107591 224776406 123.14 124.11 1994178 1684329 265.072 2382.715 134 5908 64.66 68.27 2282536 1289535 2282536 1289535 65.37 65.8 2282536 1303693 2282536 1242924 35814444 18.75 1.72 0 4.82 0 0.07 0 0.12 0 0.00 0 8.54 0 1994178 0 100 0 98.82 0 1.37 0 0.01 0 1.20 0 0.01 0 271.24 0 0.20 0 37587 0 2185015 0 105252 0 1634 0 2665 0 0 0 186538 0 147 0 0 0 1303 0 164847 0 1631 0 167928 0 86.45 0 1888926 0 28775 172698 6.001668114683 2185015.0 1994178.0 37587.0 105252.0 1634.0 2665.0 0.0 186538.0 1888926.0 91.3 1.7 4.8 0.1 0.1 0.0 8.5 86.4 50 50 50.00 38 109250750 27.6 21.5 21.7 29.2 0.0 37.8 24.3 smartseq 1467920 SRR4250330 SRP090061 SRS1698007 SRX2170331 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316549: 26Dp1_B09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316549 GSM2316549 26Dp1_B09_smart-seq 90497800 904978 2016-09-30 15:56:31 39110849 90497800 904978 2 904978 index:0,count:904978,average:50,stdev:0|index:1,count:904978,average:50,stdev:0 GSM2316549_r1 2.15 3.55 0.16 74180592 95501630 68457027 89674019 128.74 130.99 815337 698222 198.865 1775.851 78 4046 74.28 80.79 1013763 605616 1013763 605616 75.41 77.01 1013763 614815 1013763 577300 7840410 10.57 2.25 0 7.26 0 0.11 0 0.09 0 0.00 0 9.71 0 815337 0 100 0 98.38 0 1.31 0 0.01 0 1.18 0 0.01 0 191.64 0 0.22 0 20358 0 904978 0 65681 0 1004 0 790 0 0 0 87847 0 41 0 0 0 776 0 91124 0 567 0 92508 0 82.84 0 749656 0 25378 92411 3.641382299630 904978.0 815337.0 20358.0 65681.0 1004.0 790.0 0.0 87847.0 749656.0 90.1 2.2 7.3 0.1 0.1 0.0 9.7 82.8 50 50 50.00 28 45248900 26.5 22.5 22.4 28.6 0.0 36.7 25.7 smartseq 1467921 SRR4251330 SRP090061 SRS1699006 SRX2171331 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317549: 54Dn4_F04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317549 GSM2317549 54Dn4_F04_smart-seq 87901000 879010 2016-09-30 15:56:31 59034600 87901000 879010 2 879010 index:0,count:879010,average:50,stdev:0|index:1,count:879010,average:50,stdev:0 GSM2317549_r1 2.53 3.03 0.1 79210033 95173055 76304963 92149214 120.15 120.76 817737 687288 303.653 2351.564 169 2033 61.01 63.38 897620 498928 897620 498928 60.85 61.14 897620 497611 897620 481269 17951246 22.66 1.62 0 3.48 0 0.08 0 0.15 0 0.00 0 6.74 0 817737 0 100 0 98.94 0 1.40 0 0.01 0 1.23 0 0.01 0 210.96 0 0.22 0 14244 0 879010 0 30560 0 737 0 1302 0 0 0 59234 0 69 0 0 0 515 0 60772 0 670 0 62026 0 89.55 0 787177 0 19268 63768 3.309528752335 879010.0 817737.0 14244.0 30560.0 737.0 1302.0 0.0 59234.0 787177.0 93.0 1.6 3.5 0.1 0.1 0.0 6.7 89.6 50 50 50.00 38 43950500 28.0 21.1 21.2 29.7 0.0 37.7 24.3 smartseq 1467953 SRR4250331 SRP090061 SRS1698008 SRX2170332 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316550: 26Dp1_B10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316550 GSM2316550 26Dp1_B10_smart-seq 97798800 977988 2016-09-30 15:56:31 42903167 97798800 977988 2 977988 index:0,count:977988,average:50,stdev:0|index:1,count:977988,average:50,stdev:0 GSM2316550_r1 3.86 2.93 0.08 80288180 105142740 73407122 98060952 130.96 133.59 878933 731339 210.288 2008.847 69 4153 78.11 85.77 1117300 686524 1117300 686524 79.72 81.76 1117300 700690 1117300 654415 5860267 7.30 2.32 0 8.03 0 0.11 0 0.10 0 0.00 0 9.92 0 878933 0 100 0 98.35 0 1.26 0 0.01 0 1.17 0 0.01 0 185.30 0 0.23 0 22729 0 977988 0 78554 0 1055 0 977 0 0 0 97023 0 70 0 0 0 818 0 108884 0 692 0 110464 0 81.84 0 800379 0 27526 111224 4.040688803313 977988.0 878933.0 22729.0 78554.0 1055.0 977.0 0.0 97023.0 800379.0 89.9 2.3 8.0 0.1 0.1 0.0 9.9 81.8 50 50 50.00 28 48899400 26.0 23.0 22.9 28.2 0.0 36.7 25.7 smartseq 1467954 SRR4251331 SRP090061 SRS1699009 SRX2171332 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317550: 54Dn4_F05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317550 GSM2317550 54Dn4_F05_smart-seq 231826100 2318261 2016-09-30 15:56:31 154305711 231826100 2318261 2 2318261 index:0,count:2318261,average:50,stdev:0|index:1,count:2318261,average:50,stdev:0 GSM2317550_r1 2.07 3.19 0.12 210430038 258486811 200984553 248235089 122.84 123.51 2174696 1774584 287.000 2878.323 134 5879 66.24 69.41 2426655 1440497 2426655 1440497 66.42 66.76 2426655 1444335 2426655 1385438 38878406 18.48 1.62 0 4.29 0 0.09 0 0.18 0 0.00 0 5.92 0 2174696 0 100 0 98.90 0 1.40 0 0.01 0 1.21 0 0.01 0 260.80 0 0.20 0 37441 0 2318261 0 99426 0 2085 0 4213 0 0 0 137267 0 181 0 0 0 1539 0 200583 0 1771 0 204074 0 89.52 0 2075270 0 33283 212249 6.377099420124 2318261.0 2174696.0 37441.0 99426.0 2085.0 4213.0 0.0 137267.0 2075270.0 93.8 1.6 4.3 0.1 0.2 0.0 5.9 89.5 50 50 50.00 38 115913050 27.7 21.5 21.6 29.1 0.0 37.8 24.8 smartseq 1467984 SRR4250332 SRP090061 SRS1698009 SRX2170333 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316551: 26Dp1_B11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316551 GSM2316551 26Dp1_B11_smart-seq 48160800 481608 2016-09-30 15:56:31 21048883 48160800 481608 2 481608 index:0,count:481608,average:50,stdev:0|index:1,count:481608,average:50,stdev:0 GSM2316551_r1 2.35 3.65 0.11 38549977 49720709 35339590 46546424 128.98 131.71 423138 361374 202.915 1778.745 54 2251 71.43 78.24 542316 302249 542316 302249 72.77 74.6 542316 307930 542316 288178 4700881 12.19 2.23 0 7.65 0 0.13 0 0.14 0 0.00 0 11.88 0 423138 0 100 0 98.31 0 1.30 0 0.01 0 1.17 0 0.01 0 101.99 0 0.23 0 10752 0 481608 0 36851 0 618 0 656 0 0 0 57196 0 27 0 0 0 361 0 49019 0 309 0 49716 0 80.21 0 386287 0 17582 49033 2.788818109430 481608.0 423138.0 10752.0 36851.0 618.0 656.0 0.0 57196.0 386287.0 87.9 2.2 7.7 0.1 0.1 0.0 11.9 80.2 50 50 50.00 28 24080400 26.1 22.6 22.4 28.9 0.0 36.7 24.8 smartseq 1467985 SRR4251332 SRP090061 SRS1699007 SRX2171333 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317551: 54Dn4_F06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317551 GSM2317551 54Dn4_F06_smart-seq 156426900 1564269 2016-09-30 15:56:31 104909538 156426900 1564269 2 1564269 index:0,count:1564269,average:50,stdev:0|index:1,count:1564269,average:50,stdev:0 GSM2317551_r1 0.04 3.03 0.16 141030154 168192202 135683187 162802204 119.26 119.99 1460508 1199658 290.995 2454.832 165 3675 62.47 64.98 1604160 912385 1604160 912385 62.29 62.6 1604160 909787 1604160 878892 29948865 21.24 1.63 0 3.61 0 0.10 0 0.13 0 0.00 0 6.40 0 1460508 0 100 0 98.91 0 1.44 0 0.01 0 1.23 0 0.01 0 234.64 0 0.20 0 25496 0 1564269 0 56489 0 1530 0 2044 0 0 0 100187 0 133 0 0 0 925 0 129636 0 1312 0 132006 0 89.76 0 1404019 0 29038 135386 4.662373441697 1564269.0 1460508.0 25496.0 56489.0 1530.0 2044.0 0.0 100187.0 1404019.0 93.4 1.6 3.6 0.1 0.1 0.0 6.4 89.8 50 50 50.00 38 78213450 27.8 21.3 21.5 29.4 0.0 37.8 24.5 smartseq 1468016 SRR4251333 SRP090061 SRS1699008 SRX2171334 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317552: 54Dn4_F07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317552 GSM2317552 54Dn4_F07_smart-seq 47998000 479980 2016-09-30 15:56:31 32802181 47998000 479980 2 479980 index:0,count:479980,average:50,stdev:0|index:1,count:479980,average:50,stdev:0 GSM2317552_r1 1.81 3.27 0.12 43385363 54039954 41313448 51785233 124.56 125.35 447438 336527 333.271 3095.408 165 892 70.14 73.72 498825 313817 498825 313817 70.38 70.8 498825 314890 498825 301377 6919564 15.95 1.86 0 4.53 0 0.08 0 0.08 0 0.00 0 6.62 0 447438 0 100 0 98.80 0 1.47 0 0.01 0 1.26 0 0.01 0 132.92 0 0.22 0 8905 0 479980 0 21746 0 361 0 403 0 0 0 31778 0 29 0 0 0 414 0 49764 0 353 0 50560 0 88.69 0 425692 0 22564 51011 2.260725048750 479980.0 447438.0 8905.0 21746.0 361.0 403.0 0.0 31778.0 425692.0 93.2 1.9 4.5 0.1 0.1 0.0 6.6 88.7 50 50 50.00 38 23999000 27.7 21.5 21.7 29.1 0.0 37.5 24.2 smartseq 1476304 SRR4251436 SRP090061 SRS1699115 SRX2171437 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317655: 54Dp1SK_H05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317655 GSM2317655 54Dp1SK_H05_smart-seq 92456100 924561 2016-09-30 15:56:31 61642796 92456100 924561 2 924561 index:0,count:924561,average:50,stdev:0|index:1,count:924561,average:50,stdev:0 GSM2317655_r1 4.45 2.81 0.28 82907247 97275190 78591216 93360748 117.33 118.79 855319 727012 334.910 2425.526 206 1809 50.04 52.85 1001521 427963 1001521 427963 50.51 50.96 1001521 432014 1001521 412679 23868570 28.79 1.89 0 4.92 0 0.10 0 0.16 0 0.00 0 7.24 0 855319 0 100 0 98.79 0 1.45 0 0.02 0 1.20 0 0.01 0 221.89 0 0.23 0 17455 0 924561 0 45529 0 909 0 1438 0 0 0 66895 0 48 0 0 0 447 0 51341 0 893 0 52729 0 87.59 0 809790 0 19059 53063 2.784143973976 924561.0 855319.0 17455.0 45529.0 909.0 1438.0 0.0 66895.0 809790.0 92.5 1.9 4.9 0.1 0.2 0.0 7.2 87.6 50 50 50.00 38 46228050 28.3 20.6 20.8 30.3 0.0 37.8 23.6 smartseq 1476336 SRR4251437 SRP090061 SRS1699114 SRX2171438 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317656: 54Dp1SK_H06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317656 GSM2317656 54Dp1SK_H06_smart-seq 149327900 1493279 2016-09-30 15:56:31 98884029 149327900 1493279 2 1493279 index:0,count:1493279,average:50,stdev:0|index:1,count:1493279,average:50,stdev:0 GSM2317656_r1 3.01 2.35 0.29 133156339 155868105 127328246 150139345 117.06 117.92 1377839 1209042 304.126 1888.786 137 3352 49.27 51.58 1560220 678857 1560220 678857 49.86 49.97 1560220 687054 1560220 657693 39900596 29.97 1.61 0 4.13 0 0.12 0 0.14 0 0.00 0 7.46 0 1377839 0 100 0 98.99 0 1.41 0 0.01 0 1.21 0 0.01 0 316.22 0 0.21 0 24032 0 1493279 0 61640 0 1812 0 2157 0 0 0 111471 0 60 0 0 0 590 0 73310 0 1431 0 75391 0 88.14 0 1316199 0 14268 77413 5.425637790861 1493279.0 1377839.0 24032.0 61640.0 1812.0 2157.0 0.0 111471.0 1316199.0 92.3 1.6 4.1 0.1 0.1 0.0 7.5 88.1 50 50 50.00 38 74663950 28.1 20.7 20.8 30.4 0.0 37.9 23.5 smartseq 1476368 SRR4251438 SRP090061 SRS1699113 SRX2171439 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317657: 54Dp1SK_H07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317657 GSM2317657 54Dp1SK_H07_smart-seq 160178600 1601786 2016-09-30 15:56:31 105667688 160178600 1601786 2 1601786 index:0,count:1601786,average:50,stdev:0|index:1,count:1601786,average:50,stdev:0 GSM2317657_r1 2.95 2.51 0.33 145017213 158081465 138802469 152466402 109.01 109.84 1496936 1342241 306.758 1746.565 167 3758 40.61 42.47 1686189 607853 1686189 607853 41.05 41.01 1686189 614550 1686189 587030 47862876 33.00 1.54 0 4.09 0 0.15 0 0.28 0 0.00 0 6.11 0 1496936 0 100 0 98.99 0 1.43 0 0.01 0 1.22 0 0.01 0 240.27 0 0.22 0 24695 0 1601786 0 65586 0 2417 0 4548 0 0 0 97885 0 49 0 0 0 678 0 63806 0 1399 0 65932 0 89.36 0 1431350 0 16221 67118 4.137722705135 1601786.0 1496936.0 24695.0 65586.0 2417.0 4548.0 0.0 97885.0 1431350.0 93.5 1.5 4.1 0.2 0.3 0.0 6.1 89.4 50 50 50.00 38 80089300 28.4 20.6 20.8 30.1 0.0 38.0 24.7 smartseq 1477712 SRR4250462 SRP090061 SRS1698139 SRX2170463 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316681: 26Dn1_F10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316681 GSM2316681 26Dn1_F10_smart-seq 108521200 1085212 2016-09-30 15:56:31 48045074 108521200 1085212 2 1085212 index:0,count:1085212,average:50,stdev:0|index:1,count:1085212,average:50,stdev:0 GSM2316681_r1 4.83 3.32 0.15 87908891 112727855 82539689 106572179 128.23 129.12 954902 818034 200.204 1654.558 78 4350 76.12 81.33 1095621 726854 1095621 726854 77.48 78.28 1095621 739812 1095621 699558 8637281 9.83 1.93 0 5.64 0 0.10 0 0.11 0 0.00 0 11.80 0 954902 0 100 0 98.53 0 1.36 0 0.01 0 1.15 0 0.01 0 260.45 0 0.20 0 20942 0 1085212 0 61231 0 1068 0 1219 0 0 0 128023 0 81 0 0 0 700 0 96194 0 616 0 97591 0 82.35 0 893671 0 23224 97825 4.212237340682 1085212.0 954902.0 20942.0 61231.0 1068.0 1219.0 0.0 128023.0 893671.0 88.0 1.9 5.6 0.1 0.1 0.0 11.8 82.3 50 50 50.00 28 54260600 27.7 21.1 21.1 30.1 0.0 36.3 22.5 smartseq 1477713 SRR4251462 SRP090061 SRS1699139 SRX2171463 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317681: 54Dn1SK_B11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317681 GSM2317681 54Dn1SK_B11_smart-seq 129258900 1292589 2016-09-30 15:56:31 90352708 129258900 1292589 2 1292589 index:0,count:1292589,average:50,stdev:0|index:1,count:1292589,average:50,stdev:0 GSM2317681_r1 12.83 1.99 0.06 113375731 151765061 101355998 139998858 133.86 138.13 1189600 1014156 257.423 1584.976 134 3818 69.13 77.49 1615152 822341 1615152 822341 72.89 74.75 1615152 867085 1615152 793230 12987521 11.46 2.29 0 9.93 0 0.09 0 0.14 0 0.00 0 7.73 0 1189600 0 100 0 98.47 0 1.42 0 0.03 0 1.17 0 0.01 0 387.78 0 0.31 0 29650 0 1292589 0 128369 0 1220 0 1847 0 0 0 99922 0 41 0 0 0 799 0 100385 0 1258 0 102483 0 82.10 0 1061231 0 15057 104325 6.928671050010 1292589.0 1189600.0 29650.0 128369.0 1220.0 1847.0 0.0 99922.0 1061231.0 92.0 2.3 9.9 0.1 0.1 0.0 7.7 82.1 50 50 50.00 38 64629450 26.4 22.7 22.9 28.0 0.0 37.2 23.4 smartseq 1477776 SRR4251464 SRP090061 SRS1699146 SRX2171465 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317683: 54Dn1SK_C02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317683 GSM2317683 54Dn1SK_C02_smart-seq 143742000 1437420 2016-09-30 15:56:31 98601720 143742000 1437420 2 1437420 index:0,count:1437420,average:50,stdev:0|index:1,count:1437420,average:50,stdev:0 GSM2317683_r1 6.46 1.54 0.61 126548274 126894294 119009875 120710091 100.27 101.43 1333204 1246820 244.916 1101.328 134 4879 38.77 41.3 1591254 516869 1591254 516869 40.11 40.11 1591254 534692 1591254 501868 33939949 26.82 1.58 0 5.69 0 0.37 0 0.34 0 0.00 0 6.55 0 1333204 0 100 0 98.89 0 1.39 0 0.02 0 1.18 0 0.01 0 398.05 0 0.29 0 22696 0 1437420 0 81834 0 5288 0 4842 0 0 0 94086 0 41 0 0 0 233 0 42499 0 785 0 43558 0 87.06 0 1251370 0 8901 43877 4.929446129648 1437420.0 1333204.0 22696.0 81834.0 5288.0 4842.0 0.0 94086.0 1251370.0 92.7 1.6 5.7 0.4 0.3 0.0 6.5 87.1 50 50 50.00 38 71871000 28.9 20.3 20.4 30.4 0.0 37.5 24.3 smartseq 1477808 SRR4251465 SRP090061 SRS1699144 SRX2171466 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317684: 54Dn1SK_C03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317684 GSM2317684 54Dn1SK_C03_smart-seq 194375500 1943755 2016-09-30 15:56:31 133415679 194375500 1943755 2 1943755 index:0,count:1943755,average:50,stdev:0|index:1,count:1943755,average:50,stdev:0 GSM2317684_r1 7.18 2.81 0.09 169390055 226419211 153792064 210156194 133.67 136.65 1798864 1514788 227.661 1698.014 134 7307 82.77 91.4 2298921 1488852 2298921 1488852 86.18 88.39 2298921 1550278 2298921 1439937 7270286 4.29 2.11 0 8.74 0 0.09 0 0.03 0 0.00 0 7.34 0 1798864 0 100 0 98.52 0 1.43 0 0.02 0 1.17 0 0.01 0 349.88 0 0.27 0 40929 0 1943755 0 169855 0 1794 0 519 0 0 0 142578 0 42 0 0 0 966 0 162380 0 1497 0 164885 0 83.81 0 1629009 0 16671 168182 10.088297042769 1943755.0 1798864.0 40929.0 169855.0 1794.0 519.0 0.0 142578.0 1629009.0 92.5 2.1 8.7 0.1 0.0 0.0 7.3 83.8 50 50 50.00 38 97187750 28.2 20.9 21.1 29.8 0.0 37.5 23.9 smartseq 1477840 SRR4251466 SRP090061 SRS1699141 SRX2171467 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317685: 54Dn1SK_C04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317685 GSM2317685 54Dn1SK_C04_smart-seq 189919100 1899191 2016-09-30 15:56:31 127632217 189919100 1899191 2 1899191 index:0,count:1899191,average:50,stdev:0|index:1,count:1899191,average:50,stdev:0 GSM2317685_r1 6.46 2.55 0.13 164163618 198379815 156403705 191864675 120.84 122.67 1777135 1726278 188.197 648.985 100 9252 50.37 53.0 2090673 895217 2090673 895217 50.73 51.7 2090673 901494 2090673 873208 46229842 28.16 1.49 0 4.64 0 0.14 0 0.22 0 0.00 0 6.07 0 1777135 0 100 0 98.57 0 1.52 0 0.03 0 1.18 0 0.01 0 284.88 0 0.23 0 28204 0 1899191 0 88104 0 2608 0 4123 0 0 0 115325 0 56 0 0 0 292 0 30963 0 1119 0 32430 0 88.93 0 1689031 0 6295 32388 5.145035742653 1899191.0 1777135.0 28204.0 88104.0 2608.0 4123.0 0.0 115325.0 1689031.0 93.6 1.5 4.6 0.1 0.2 0.0 6.1 88.9 50 50 50.00 38 94959550 30.2 18.7 18.9 32.2 0.0 37.7 24.7 smartseq 1477872 SRR4251467 SRP090061 SRS1699145 SRX2171468 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317686: 54Dn1SK_C05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317686 GSM2317686 54Dn1SK_C05_smart-seq 206636100 2066361 2016-09-30 15:56:31 140117744 206636100 2066361 2 2066361 index:0,count:2066361,average:50,stdev:0|index:1,count:2066361,average:50,stdev:0 GSM2317686_r1 3.91 3.3 0.11 183565682 241698156 173371213 230358722 131.67 132.87 1943477 1658910 215.230 1655.811 116 8215 83.74 88.81 2215881 1627391 2215881 1627391 84.51 85.85 2215881 1642431 2215881 1573253 9755257 5.31 1.53 0 5.37 0 0.06 0 0.07 0 0.00 0 5.82 0 1943477 0 100 0 98.80 0 1.37 0 0.01 0 1.16 0 0.01 0 232.47 0 0.21 0 31653 0 2066361 0 110957 0 1211 0 1374 0 0 0 120299 0 198 0 0 0 1105 0 168807 0 1105 0 171215 0 88.68 0 1832520 0 15268 175817 11.515391668850 2066361.0 1943477.0 31653.0 110957.0 1211.0 1374.0 0.0 120299.0 1832520.0 94.1 1.5 5.4 0.1 0.1 0.0 5.8 88.7 50 50 50.00 38 103318050 29.4 19.8 20.1 30.7 0.0 37.6 24.6 smartseq 1477904 SRR4250468 SRP090061 SRS1698145 SRX2170469 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316687: 26Dn1_G05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316687 GSM2316687 26Dn1_G05_smart-seq 80011300 800113 2016-09-30 15:56:31 36307833 80011300 800113 2 800113 index:0,count:800113,average:50,stdev:0|index:1,count:800113,average:50,stdev:0 GSM2316687_r1 1.99 3.76 0.18 64629177 81300787 60208046 76574658 125.8 127.18 695665 568099 236.854 2129.386 54 2664 74.13 79.81 825784 515710 825784 515710 75.11 76.18 825784 522543 825784 492288 7105509 10.99 2.11 0 6.19 0 0.12 0 0.11 0 0.00 0 12.83 0 695665 0 100 0 98.35 0 1.31 0 0.01 0 1.17 0 0.01 0 221.57 0 0.21 0 16910 0 800113 0 49490 0 969 0 843 0 0 0 102636 0 76 0 0 0 590 0 78814 0 519 0 79999 0 80.76 0 646175 0 21092 80809 3.831263038119 800113.0 695665.0 16910.0 49490.0 969.0 843.0 0.0 102636.0 646175.0 86.9 2.1 6.2 0.1 0.1 0.0 12.8 80.8 50 50 50.00 28 40005650 26.7 22.1 22.1 29.2 0.0 36.2 22.0 smartseq 1477905 SRR4251468 SRP090061 SRS1699142 SRX2171469 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317687: 54Dn1SK_C06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317687 GSM2317687 54Dn1SK_C06_smart-seq 138350000 1383500 2016-09-30 15:56:31 95223950 138350000 1383500 2 1383500 index:0,count:1383500,average:50,stdev:0|index:1,count:1383500,average:50,stdev:0 GSM2317687_r1 2.12 3.05 0.07 122358376 146498352 117177723 141500540 119.73 120.76 1290239 1116740 238.558 1839.652 134 4501 54.48 56.95 1455345 702921 1455345 702921 54.47 54.89 1455345 702820 1455345 677483 32568686 26.62 1.72 0 4.05 0 0.09 0 0.12 0 0.00 0 6.53 0 1290239 0 100 0 98.76 0 1.50 0 0.03 0 1.21 0 0.01 0 355.76 0 0.26 0 23749 0 1383500 0 55981 0 1258 0 1604 0 0 0 90399 0 147 0 0 0 701 0 88179 0 1547 0 90574 0 89.21 0 1234258 0 23775 91133 3.833144058885 1383500.0 1290239.0 23749.0 55981.0 1258.0 1604.0 0.0 90399.0 1234258.0 93.3 1.7 4.0 0.1 0.1 0.0 6.5 89.2 50 50 50.00 38 69175000 29.1 20.1 20.3 30.5 0.0 37.4 24.0 smartseq 1477936 SRR4251469 SRP090061 SRS1699143 SRX2171470 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317688: 54Dn1SK_C07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317688 GSM2317688 54Dn1SK_C07_smart-seq 263533000 2635330 2016-09-30 15:56:31 179175364 263533000 2635330 2 2635330 index:0,count:2635330,average:50,stdev:0|index:1,count:2635330,average:50,stdev:0 GSM2317688_r1 5.09 3.52 0.15 230107324 283408098 215258486 267768791 123.16 124.39 2464336 2170851 207.904 1457.553 111 11087 61.83 66.21 2885757 1523657 2885757 1523657 63.76 64.04 2885757 1571277 2885757 1473679 43418887 18.87 2.02 0 6.19 0 0.10 0 0.10 0 0.00 0 6.29 0 2464336 0 100 0 98.76 0 1.43 0 0.02 0 1.21 0 0.01 0 338.83 0 0.24 0 53187 0 2635330 0 163241 0 2758 0 2504 0 0 0 165732 0 161 0 0 0 1280 0 176077 0 2195 0 179713 0 87.32 0 2301095 0 19752 182499 9.239520048603 2635330.0 2464336.0 53187.0 163241.0 2758.0 2504.0 0.0 165732.0 2301095.0 93.5 2.0 6.2 0.1 0.1 0.0 6.3 87.3 50 50 50.00 38 131766500 29.0 20.3 20.4 30.3 0.0 37.6 24.7 smartseq 1479408 SRR4251497 SRP090061 SRS1699176 SRX2171498 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317716: 54Dn1SK_F02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317716 GSM2317716 54Dn1SK_F02_smart-seq 230361700 2303617 2016-09-30 15:56:31 155641906 230361700 2303617 2 2303617 index:0,count:2303617,average:50,stdev:0|index:1,count:2303617,average:50,stdev:0 GSM2317716_r1 0.77 1.53 0.27 194257016 220117376 188022734 214749507 113.31 114.21 2092498 1981495 186.555 874.603 100 11310 61.21 63.3 2304917 1280821 2304917 1280821 60.99 61.88 2304917 1276120 2304917 1252157 46129995 23.75 1.43 0 3.00 0 0.07 0 0.15 0 0.00 0 8.94 0 2092498 0 100 0 98.83 0 1.41 0 0.01 0 1.18 0 0.01 0 331.72 0 0.20 0 33033 0 2303617 0 69050 0 1721 0 3551 0 0 0 205847 0 190 0 0 0 1072 0 70478 0 1654 0 73394 0 87.84 0 2023448 0 4575 73699 16.109071038251 2303617.0 2092498.0 33033.0 69050.0 1721.0 3551.0 0.0 205847.0 2023448.0 90.8 1.4 3.0 0.1 0.2 0.0 8.9 87.8 50 50 50.00 38 115180850 29.1 19.8 19.8 31.4 0.0 37.6 23.2 smartseq 1479472 SRR4251499 SRP090061 SRS1699175 SRX2171500 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317718: 54Dn1SK_F04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317718 GSM2317718 54Dn1SK_F04_smart-seq 388736900 3887369 2016-09-30 15:56:31 259879825 388736900 3887369 2 3887369 index:0,count:3887369,average:50,stdev:0|index:1,count:3887369,average:50,stdev:0 GSM2317718_r1 7.33 2.53 0.12 333871105 403116497 311717565 380368233 120.74 122.02 3651418 3399297 171.911 823.845 100 22246 59.89 64.32 4321376 2186923 4321376 2186923 62.31 62.68 4321376 2275147 4321376 2131171 67132651 20.11 1.87 0 6.46 0 0.14 0 0.24 0 0.00 0 5.69 0 3651418 0 100 0 98.77 0 1.42 0 0.02 0 1.19 0 0.01 0 451.44 0 0.22 0 72607 0 3887369 0 251125 0 5480 0 9254 0 0 0 221217 0 144 0 0 0 1067 0 172058 0 3180 0 176449 0 87.47 0 3400293 0 9345 177591 19.003852327448 3887369.0 3651418.0 72607.0 251125.0 5480.0 9254.0 0.0 221217.0 3400293.0 93.9 1.9 6.5 0.1 0.2 0.0 5.7 87.5 50 50 50.00 38 194368450 30.0 19.4 19.5 31.1 0.0 37.8 26.0 smartseq 1482768 SRR4251500 SRP090061 SRS1699179 SRX2171501 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317719: 54Dn1SK_F05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317719 GSM2317719 54Dn1SK_F05_smart-seq 321513200 3215132 2016-09-30 15:56:31 216310109 321513200 3215132 2 3215132 index:0,count:3215132,average:50,stdev:0|index:1,count:3215132,average:50,stdev:0 GSM2317719_r1 8.13 2.83 0.2 285196873 346675276 269567803 332125829 121.56 123.21 3049510 2783919 192.963 1174.079 116 15264 56.45 59.82 3615589 1721358 3615589 1721358 57.67 58.29 3615589 1758562 3615589 1677215 68088471 23.87 1.54 0 5.35 0 0.25 0 0.24 0 0.00 0 4.66 0 3049510 0 100 0 98.93 0 1.39 0 0.01 0 1.23 0 0.01 0 312.82 0 0.23 0 49627 0 3215132 0 171915 0 8007 0 7671 0 0 0 149944 0 73 0 0 0 886 0 166937 0 2157 0 170053 0 89.50 0 2877595 0 16110 171023 10.615952824333 3215132.0 3049510.0 49627.0 171915.0 8007.0 7671.0 0.0 149944.0 2877595.0 94.8 1.5 5.3 0.2 0.2 0.0 4.7 89.5 50 50 50.00 38 160756600 29.5 19.9 20.1 30.5 0.0 37.8 25.9 smartseq 1483024 SRR4250508 SRP090061 SRS1698185 SRX2170509 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316727: 54Dp1_C01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316727 GSM2316727 54Dp1_C01_smart-seq 49089600 490896 2016-09-30 15:56:31 23317455 49089600 490896 2 490896 index:0,count:490896,average:50,stdev:0|index:1,count:490896,average:50,stdev:0 GSM2316727_r1 7.46 3.31 0.12 42928674 58692021 39589718 55261100 136.72 139.58 451392 345845 299.004 3594.323 78 1211 82.43 89.66 572944 372091 572944 372091 83.09 85.75 572944 375060 572944 355873 2335552 5.44 1.83 0 7.41 0 0.08 0 0.04 0 0.00 0 7.93 0 451392 0 100 0 98.61 0 1.23 0 0.01 0 1.15 0 0.01 0 126.23 0 0.22 0 8960 0 490896 0 36399 0 384 0 182 0 0 0 38938 0 55 0 0 0 418 0 54899 0 253 0 55625 0 84.54 0 414993 0 24058 56054 2.329952614515 490896.0 451392.0 8960.0 36399.0 384.0 182.0 0.0 38938.0 414993.0 92.0 1.8 7.4 0.1 0.0 0.0 7.9 84.5 50 50 50.00 28 24544800 26.4 22.8 22.9 27.9 0.0 36.1 23.5 smartseq 1483025 SRR4251508 SRP090061 SRS1699185 SRX2171509 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317727: 54Dn1SK_G02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317727 GSM2317727 54Dn1SK_G02_smart-seq 165155500 1651555 2016-09-30 15:56:31 112949274 165155500 1651555 2 1651555 index:0,count:1651555,average:50,stdev:0|index:1,count:1651555,average:50,stdev:0 GSM2317727_r1 8.44 1.89 0.17 139202313 174548520 127019485 160907660 125.39 126.68 1487747 1351611 212.343 1024.209 134 6824 71.96 79.05 1839465 1070594 1839465 1070594 76.05 76.95 1839465 1131375 1839465 1042172 14285783 10.26 2.19 0 8.08 0 0.12 0 0.10 0 0.00 0 9.70 0 1487747 0 100 0 98.38 0 1.46 0 0.02 0 1.21 0 0.01 0 283.12 0 0.26 0 36160 0 1651555 0 133431 0 1956 0 1611 0 0 0 160241 0 59 0 0 0 322 0 83046 0 1448 0 84875 0 82.00 0 1354316 0 4981 86791 17.424412768520 1651555.0 1487747.0 36160.0 133431.0 1956.0 1611.0 0.0 160241.0 1354316.0 90.1 2.2 8.1 0.1 0.1 0.0 9.7 82.0 50 50 50.00 38 82577750 27.7 21.1 21.2 30.0 0.0 37.5 23.1 smartseq 1483056 SRR4251509 SRP090061 SRS1699186 SRX2171510 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317728: 54Dn1SK_G03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317728 GSM2317728 54Dn1SK_G03_smart-seq 133528400 1335284 2016-09-30 15:56:31 92173656 133528400 1335284 2 1335284 index:0,count:1335284,average:50,stdev:0|index:1,count:1335284,average:50,stdev:0 GSM2317728_r1 2.11 3.14 0.16 120214573 143524603 115621793 139077689 119.39 120.29 1255075 1093815 260.708 1820.947 133 3876 53.83 56.02 1398397 675587 1398397 675587 53.72 54.05 1398397 674238 1398397 651749 31710021 26.38 1.62 0 3.68 0 0.08 0 0.12 0 0.00 0 5.80 0 1255075 0 100 0 98.95 0 1.52 0 0.02 0 1.22 0 0.01 0 253.00 0 0.26 0 21594 0 1335284 0 49196 0 1051 0 1665 0 0 0 77493 0 55 0 0 0 633 0 78996 0 1249 0 80933 0 90.31 0 1205879 0 25291 80659 3.189237278083 1335284.0 1255075.0 21594.0 49196.0 1051.0 1665.0 0.0 77493.0 1205879.0 94.0 1.6 3.7 0.1 0.1 0.0 5.8 90.3 50 50 50.00 38 66764200 29.3 20.0 20.2 30.5 0.0 37.5 24.6 smartseq 1483409 SRR4250514 SRP090061 SRS1698192 SRX2170515 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316733: 54Dp1_C07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316733 GSM2316733 54Dp1_C07_smart-seq 115436100 1154361 2016-09-30 15:56:31 52114180 115436100 1154361 2 1154361 index:0,count:1154361,average:50,stdev:0|index:1,count:1154361,average:50,stdev:0 GSM2316733_r1 4.22 3.14 0.2 97061968 127340003 90723296 120828941 131.19 133.18 1047045 870008 224.618 2143.211 100 4284 76.99 82.61 1261669 806103 1261669 806103 77.06 78.79 1261669 806815 1261669 768823 9033383 9.31 1.81 0 6.18 0 0.15 0 0.06 0 0.00 0 9.08 0 1047045 0 100 0 98.66 0 1.25 0 0.01 0 1.17 0 0.01 0 277.05 0 0.20 0 20919 0 1154361 0 71304 0 1774 0 693 0 0 0 104849 0 83 0 0 0 913 0 109090 0 593 0 110679 0 84.53 0 975741 0 29732 111548 3.751782591148 1154361.0 1047045.0 20919.0 71304.0 1774.0 693.0 0.0 104849.0 975741.0 90.7 1.8 6.2 0.2 0.1 0.0 9.1 84.5 50 50 50.00 28 57718050 27.4 21.8 21.7 29.1 0.0 36.3 24.1 smartseq 1483410 SRR4251514 SRP090061 SRS1699193 SRX2171515 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317733: 54Dn1SK_G08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317733 GSM2317733 54Dn1SK_G08_smart-seq 155356200 1553562 2016-09-30 15:56:31 106592551 155356200 1553562 2 1553562 index:0,count:1553562,average:50,stdev:0|index:1,count:1553562,average:50,stdev:0 GSM2317733_r1 14.54 0.78 0.27 133318934 156342911 117218659 146792608 117.27 125.23 1420081 1387281 200.701 845.848 134 7560 56.6 64.58 2305981 803796 2305981 803796 58.5 63.7 2305981 830725 2305981 792938 21662692 16.25 1.96 0 11.29 0 0.30 0 0.26 0 0.00 0 8.03 0 1420081 0 100 0 98.43 0 1.30 0 0.02 0 1.13 0 0.01 0 199.74 0 0.32 0 30431 0 1553562 0 175363 0 4643 0 4077 0 0 0 124761 0 0 0 0 0 90 0 13503 0 921 0 14514 0 80.12 0 1244718 0 1977 14857 7.514921598381 1553562.0 1420081.0 30431.0 175363.0 4643.0 4077.0 0.0 124761.0 1244718.0 91.4 2.0 11.3 0.3 0.3 0.0 8.0 80.1 50 50 50.00 38 77678100 27.2 21.8 22.0 29.0 0.0 37.5 23.5 smartseq 1483856 SRR4251522 SRP090061 SRS1699197 SRX2171523 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317741: 54Dn1SK_H05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317741 GSM2317741 54Dn1SK_H05_smart-seq 253218800 2532188 2016-09-30 15:56:31 171927310 253218800 2532188 2 2532188 index:0,count:2532188,average:50,stdev:0|index:1,count:2532188,average:50,stdev:0 GSM2317741_r1 0.71 3.14 0.05 225761633 272692453 217503528 264560124 120.79 121.63 2379227 2134805 209.154 1424.287 134 10403 68.07 70.73 2634168 1619535 2634168 1619535 67.98 68.61 2634168 1617492 2634168 1571013 39610844 17.55 1.23 0 3.53 0 0.16 0 0.16 0 0.00 0 5.72 0 2379227 0 100 0 98.92 0 1.44 0 0.01 0 1.22 0 0.01 0 364.64 0 0.22 0 31153 0 2532188 0 89345 0 4064 0 3938 0 0 0 144959 0 62 0 0 0 993 0 161027 0 1360 0 163442 0 90.43 0 2289882 0 8843 166334 18.809679972860 2532188.0 2379227.0 31153.0 89345.0 4064.0 3938.0 0.0 144959.0 2289882.0 94.0 1.2 3.5 0.2 0.2 0.0 5.7 90.4 50 50 50.00 38 126609400 28.3 20.9 21.1 29.6 0.0 37.6 25.1 smartseq 1483889 SRR4250523 SRP090061 SRS1698200 SRX2170524 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316742: 54Dp1_D05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316742 GSM2316742 54Dp1_D05_smart-seq 185907100 1859071 2016-09-30 15:56:31 81824868 185907100 1859071 2 1859071 index:0,count:1859071,average:50,stdev:0|index:1,count:1859071,average:50,stdev:0 GSM2316742_r1 9.04 3.28 0.17 161310473 217583544 151109160 206567269 134.88 136.7 1726284 1435775 223.322 2335.713 88 6825 82.16 87.96 2042488 1418298 2042488 1418298 82.9 84.67 2042488 1431024 2042488 1365261 9669202 5.99 1.65 0 6.12 0 0.09 0 0.04 0 0.00 0 7.01 0 1726284 0 100 0 98.76 0 1.26 0 0.01 0 1.16 0 0.01 0 290.99 0 0.21 0 30742 0 1859071 0 113862 0 1711 0 824 0 0 0 130252 0 98 0 0 0 1101 0 177066 0 913 0 179178 0 86.73 0 1612422 0 36614 182842 4.993772873764 1859071.0 1726284.0 30742.0 113862.0 1711.0 824.0 0.0 130252.0 1612422.0 92.9 1.7 6.1 0.1 0.0 0.0 7.0 86.7 50 50 50.00 28 92953550 28.1 21.2 21.3 29.3 0.0 36.7 25.0 smartseq 1483890 SRR4251523 SRP090061 SRS1699202 SRX2171524 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317742: 54Dn1SK_H06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317742 GSM2317742 54Dn1SK_H06_smart-seq 194841900 1948419 2016-09-30 15:56:31 133006689 194841900 1948419 2 1948419 index:0,count:1948419,average:50,stdev:0|index:1,count:1948419,average:50,stdev:0 GSM2317742_r1 0.84 2.93 0.32 171900811 205947909 162814116 197251565 119.81 121.15 1817861 1644105 215.581 1326.780 116 7518 62.8 66.43 2104134 1141579 2104134 1141579 63.75 64.49 2104134 1158952 2104134 1108254 34149994 19.87 1.57 0 5.10 0 0.15 0 0.18 0 0.00 0 6.37 0 1817861 0 100 0 98.73 0 1.46 0 0.01 0 1.20 0 0.01 0 269.78 0 0.22 0 30533 0 1948419 0 99423 0 2936 0 3546 0 0 0 124076 0 201 0 0 0 822 0 104018 0 1387 0 106428 0 88.20 0 1718438 0 8150 107772 13.223558282209 1948419.0 1817861.0 30533.0 99423.0 2936.0 3546.0 0.0 124076.0 1718438.0 93.3 1.6 5.1 0.2 0.2 0.0 6.4 88.2 50 50 50.00 38 97420950 28.9 20.1 20.4 30.6 0.0 37.6 24.5 smartseq 1483920 SRR4250524 SRP090061 SRS1698202 SRX2170525 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316743: 54Dp1_D06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316743 GSM2316743 54Dp1_D06_smart-seq 152389500 1523895 2016-09-30 15:56:31 67893455 152389500 1523895 2 1523895 index:0,count:1523895,average:50,stdev:0|index:1,count:1523895,average:50,stdev:0 GSM2316743_r1 6.47 2.82 0.11 130190878 171692782 122107940 163445054 131.88 133.85 1395823 1151945 226.011 2256.744 78 5348 78.26 83.67 1664706 1092310 1664706 1092310 78.52 80.25 1664706 1095993 1664706 1047714 10604981 8.15 1.64 0 5.92 0 0.08 0 0.08 0 0.00 0 8.25 0 1395823 0 100 0 98.70 0 1.27 0 0.01 0 1.15 0 0.01 0 219.44 0 0.21 0 25061 0 1523895 0 90258 0 1181 0 1168 0 0 0 125723 0 124 0 0 0 1104 0 148808 0 889 0 150925 0 85.67 0 1305565 0 33533 153310 4.571914233740 1523895.0 1395823.0 25061.0 90258.0 1181.0 1168.0 0.0 125723.0 1305565.0 91.6 1.6 5.9 0.1 0.1 0.0 8.3 85.7 50 50 50.00 28 76194750 27.8 21.6 21.6 29.0 0.0 36.7 24.8 smartseq 1483921 SRR4251524 SRP090061 SRS1699199 SRX2171525 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317743: 54Dn1SK_H07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317743 GSM2317743 54Dn1SK_H07_smart-seq 244582300 2445823 2016-09-30 15:56:31 165716183 244582300 2445823 2 2445823 index:0,count:2445823,average:50,stdev:0|index:1,count:2445823,average:50,stdev:0 GSM2317743_r1 5.35 1.91 0.15 217602816 263224104 206867217 251549170 120.97 121.6 2302139 2151603 196.985 959.715 116 11483 59.61 62.81 2608353 1372202 2608353 1372202 60.97 61.01 2608353 1403701 2608353 1332801 48744428 22.40 1.66 0 4.80 0 0.08 0 0.15 0 0.00 0 5.65 0 2302139 0 100 0 98.89 0 1.49 0 0.02 0 1.21 0 0.01 0 400.23 0 0.23 0 40483 0 2445823 0 117488 0 1885 0 3581 0 0 0 138218 0 24 0 0 0 840 0 93852 0 2263 0 96979 0 89.32 0 2184651 0 5383 97005 18.020620471856 2445823.0 2302139.0 40483.0 117488.0 1885.0 3581.0 0.0 138218.0 2184651.0 94.1 1.7 4.8 0.1 0.1 0.0 5.7 89.3 50 50 50.00 38 122291150 28.9 20.4 20.6 30.1 0.0 37.7 25.4 smartseq 1483953 SRR4250525 SRP090061 SRS1698201 SRX2170526 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316744: 54Dp1_D07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316744 GSM2316744 54Dp1_D07_smart-seq 110058100 1100581 2016-09-30 15:56:31 48711807 110058100 1100581 2 1100581 index:0,count:1100581,average:50,stdev:0|index:1,count:1100581,average:50,stdev:0 GSM2316744_r1 3.88 3.28 0.22 83127375 108149091 76702977 101525223 130.1 132.36 910886 792358 187.872 1556.280 100 4882 88.5 96.31 1128653 806161 1128653 806161 89.49 92.68 1128653 815139 1128653 775839 674105 0.81 1.73 0 6.71 0 0.09 0 0.02 0 0.00 0 17.13 0 910886 0 100 0 98.59 0 1.28 0 0.01 0 1.16 0 0.01 0 264.14 0 0.19 0 19091 0 1100581 0 73806 0 942 0 179 0 0 0 188574 0 62 0 0 0 597 0 87489 0 488 0 88636 0 76.06 0 837080 0 13021 89198 6.850318715920 1100581.0 910886.0 19091.0 73806.0 942.0 179.0 0.0 188574.0 837080.0 82.8 1.7 6.7 0.1 0.0 0.0 17.1 76.1 50 50 50.00 28 55029050 27.7 21.0 20.2 31.0 0.0 36.5 22.1 smartseq 1483954 SRR4251525 SRP090061 SRS1699200 SRX2171526 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317744: 54Dn1SK_H08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317744 GSM2317744 54Dn1SK_H08_smart-seq 181651100 1816511 2016-09-30 15:56:31 126803178 181651100 1816511 2 1816511 index:0,count:1816511,average:50,stdev:0|index:1,count:1816511,average:50,stdev:0 GSM2317744_r1 6.87 2.23 0.03 162562687 222005712 144237332 202375228 136.57 140.31 1701606 1354782 248.132 1959.676 134 5701 84.0 94.85 2292755 1429361 2292755 1429361 87.32 90.35 2292755 1485905 2292755 1361468 3666729 2.26 2.27 0 10.72 0 0.07 0 0.02 0 0.00 0 6.23 0 1701606 0 100 0 98.52 0 1.30 0 0.01 0 1.15 0 0.00 0 284.32 0 0.28 0 41178 0 1816511 0 194691 0 1230 0 440 0 0 0 113235 0 151 0 0 0 1203 0 210513 0 1220 0 213087 0 82.96 0 1506915 0 19605 218360 11.137975006376 1816511.0 1701606.0 41178.0 194691.0 1230.0 440.0 0.0 113235.0 1506915.0 93.7 2.3 10.7 0.1 0.0 0.0 6.2 83.0 50 50 50.00 38 90825550 26.2 23.1 23.4 27.2 0.0 37.3 24.4 smartseq 1483987 SRR4250526 SRP090061 SRS1698203 SRX2170527 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316745: 54Dp1_D08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316745 GSM2316745 54Dp1_D08_smart-seq 192335500 1923355 2016-09-30 15:56:31 85753362 192335500 1923355 2 1923355 index:0,count:1923355,average:50,stdev:0|index:1,count:1923355,average:50,stdev:0 GSM2316745_r1 7.69 3.17 0.12 163711808 217350334 153648773 207111723 132.76 134.8 1765452 1498180 206.696 2016.555 100 7753 79.09 84.5 2097956 1396225 2097956 1396225 79.67 81.32 2097956 1406562 2097956 1343737 13016588 7.95 1.65 0 5.88 0 0.07 0 0.05 0 0.00 0 8.09 0 1765452 0 100 0 98.71 0 1.26 0 0.01 0 1.15 0 0.01 0 314.73 0 0.21 0 31655 0 1923355 0 113018 0 1433 0 925 0 0 0 155545 0 121 0 0 0 1246 0 170791 0 861 0 173019 0 85.91 0 1652434 0 32870 176573 5.371858837846 1923355.0 1765452.0 31655.0 113018.0 1433.0 925.0 0.0 155545.0 1652434.0 91.8 1.6 5.9 0.1 0.0 0.0 8.1 85.9 50 50 50.00 28 96167750 28.3 21.1 21.2 29.4 0.0 36.7 25.0 smartseq 1483988 SRR4251526 SRP090061 SRS1699203 SRX2171527 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317745: 54Dn1SK_H09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317745 GSM2317745 54Dn1SK_H09_smart-seq 173548900 1735489 2016-09-30 15:56:31 119237649 173548900 1735489 2 1735489 index:0,count:1735489,average:50,stdev:0|index:1,count:1735489,average:50,stdev:0 GSM2317745_r1 4.53 2.9 0.19 156965413 197513905 147657724 187629791 125.83 127.07 1640668 1404661 235.601 1710.999 134 5817 70.1 74.62 1913900 1150115 1913900 1150115 71.4 72.07 1913900 1171410 1913900 1110842 23697669 15.10 1.56 0 5.72 0 0.06 0 0.10 0 0.00 0 5.31 0 1640668 0 100 0 98.80 0 1.43 0 0.01 0 1.19 0 0.01 0 249.91 0 0.24 0 27146 0 1735489 0 99295 0 1016 0 1705 0 0 0 92100 0 25 0 0 0 955 0 144990 0 1457 0 147427 0 88.81 0 1541373 0 12684 149777 11.808341217282 1735489.0 1640668.0 27146.0 99295.0 1016.0 1705.0 0.0 92100.0 1541373.0 94.5 1.6 5.7 0.1 0.1 0.0 5.3 88.8 50 50 50.00 38 86774450 27.3 22.0 22.2 28.4 0.0 37.5 25.0 smartseq 1484019 SRR4250527 SRP090061 SRS1698205 SRX2170528 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316746: 54Dp1_D09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316746 GSM2316746 54Dp1_D09_smart-seq 114696600 1146966 2016-09-30 15:56:31 50182825 114696600 1146966 2 1146966 index:0,count:1146966,average:50,stdev:0|index:1,count:1146966,average:50,stdev:0 GSM2316746_r1 4.23 3.11 0.17 95722354 124464824 89384644 117981191 130.03 131.99 1030201 877504 213.045 1831.294 100 4382 75.64 81.23 1243447 779236 1243447 779236 76.31 77.82 1243447 786115 1243447 746560 9770658 10.21 1.66 0 6.18 0 0.09 0 0.08 0 0.00 0 10.01 0 1030201 0 100 0 98.75 0 1.29 0 0.01 0 1.16 0 0.01 0 229.39 0 0.20 0 19091 0 1146966 0 70888 0 1001 0 944 0 0 0 114820 0 110 0 0 0 778 0 96609 0 514 0 98011 0 83.64 0 959313 0 22772 99219 4.357061303355 1146966.0 1030201.0 19091.0 70888.0 1001.0 944.0 0.0 114820.0 959313.0 89.8 1.7 6.2 0.1 0.1 0.0 10.0 83.6 50 50 50.00 28 57348300 28.2 21.0 20.8 30.1 0.0 36.6 23.7 smartseq 1484020 SRR4251527 SRP090061 SRS1699204 SRX2171528 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317746: 54Dn1SK_H10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317746 GSM2317746 54Dn1SK_H10_smart-seq 137525100 1375251 2016-09-30 15:56:31 95645220 137525100 1375251 2 1375251 index:0,count:1375251,average:50,stdev:0|index:1,count:1375251,average:50,stdev:0 GSM2317746_r1 2.87 2.58 0.18 122697851 143945459 115848630 137973220 117.32 119.1 1280694 1147934 251.922 1781.289 137 4169 54.38 57.67 1499617 696426 1499617 696426 55.11 55.82 1499617 705781 1499617 674016 30418324 24.79 1.62 0 5.32 0 0.13 0 0.17 0 0.00 0 6.58 0 1280694 0 100 0 98.90 0 1.43 0 0.01 0 1.19 0 0.01 0 412.58 0 0.25 0 22285 0 1375251 0 73144 0 1775 0 2278 0 0 0 90504 0 105 0 0 0 724 0 69599 0 1395 0 71823 0 87.81 0 1207550 0 10284 74536 7.247763516142 1375251.0 1280694.0 22285.0 73144.0 1775.0 2278.0 0.0 90504.0 1207550.0 93.1 1.6 5.3 0.1 0.2 0.0 6.6 87.8 50 50 50.00 38 68762550 27.7 21.3 21.5 29.5 0.0 37.4 24.1 smartseq 1484052 SRR4250528 SRP090061 SRS1698204 SRX2170529 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316747: 54Dp1_D10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316747 GSM2316747 54Dp1_D10_smart-seq 82929300 829293 2016-09-30 15:56:31 37806161 82929300 829293 2 829293 index:0,count:829293,average:50,stdev:0|index:1,count:829293,average:50,stdev:0 GSM2316747_r1 7.55 3.08 0.15 70844050 95964240 65732362 90673282 135.46 137.94 755399 613226 237.510 2412.791 89 2763 79.14 85.54 932848 597794 932848 597794 79.93 82.09 932848 603777 932848 573683 5520199 7.79 1.76 0 6.82 0 0.08 0 0.06 0 0.00 0 8.77 0 755399 0 100 0 98.65 0 1.25 0 0.01 0 1.15 0 0.01 0 199.03 0 0.23 0 14637 0 829293 0 56536 0 663 0 515 0 0 0 72716 0 59 0 0 0 590 0 83909 0 452 0 85010 0 84.27 0 698863 0 28363 85812 3.025490956528 829293.0 755399.0 14637.0 56536.0 663.0 515.0 0.0 72716.0 698863.0 91.1 1.8 6.8 0.1 0.1 0.0 8.8 84.3 50 50 50.00 28 41464650 27.2 22.1 22.2 28.5 0.0 36.7 24.5 smartseq 1484053 SRR4251528 SRP090061 SRS1699208 SRX2171529 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317747: 54Dn1SK_H11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX-|days in culture;;54|source_name;;cultured embryonic stem cells|viral barcoded;;viral_barcoded GEO Accession;;GSM2317747 GSM2317747 54Dn1SK_H11_smart-seq 185502800 1855028 2016-09-30 15:56:31 128237609 185502800 1855028 2 1855028 index:0,count:1855028,average:50,stdev:0|index:1,count:1855028,average:50,stdev:0 GSM2317747_r1 3.65 2.54 0.09 161648117 203614704 152915644 194904149 125.96 127.46 1710377 1464981 233.203 1887.765 113 5815 70.91 75.07 2009314 1212809 2009314 1212809 70.95 72.05 2009314 1213434 2009314 1164023 23940630 14.81 1.80 0 5.12 0 0.10 0 0.13 0 0.00 0 7.57 0 1710377 0 100 0 98.53 0 1.44 0 0.01 0 1.19 0 0.01 0 247.34 0 0.25 0 33312 0 1855028 0 94901 0 1868 0 2414 0 0 0 140369 0 127 0 0 0 925 0 141253 0 1188 0 143493 0 87.09 0 1615476 0 11535 145695 12.630689206762 1855028.0 1710377.0 33312.0 94901.0 1868.0 2414.0 0.0 140369.0 1615476.0 92.2 1.8 5.1 0.1 0.1 0.0 7.6 87.1 50 50 50.00 38 92751400 27.7 21.2 21.4 29.7 0.0 37.4 23.6 smartseq 1484084 SRR4250529 SRP090061 SRS1698206 SRX2170530 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316748: 54Dp1_D11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316748 GSM2316748 54Dp1_D11_smart-seq 57435800 574358 2016-09-30 15:56:31 26494329 57435800 574358 2 574358 index:0,count:574358,average:50,stdev:0|index:1,count:574358,average:50,stdev:0 GSM2316748_r1 5.04 3.26 0.19 47748480 62016793 44479557 58853166 129.88 132.32 507059 405532 255.298 2939.335 81 1613 77.27 83.18 623576 391782 623576 391782 77.59 79.68 623576 393442 623576 375287 4221063 8.84 1.80 0 6.28 0 0.14 0 0.07 0 0.00 0 11.50 0 507059 0 100 0 98.60 0 1.22 0 0.01 0 1.15 0 0.01 0 147.69 0 0.25 0 10338 0 574358 0 36048 0 806 0 426 0 0 0 66067 0 39 0 0 0 405 0 56338 0 312 0 57094 0 82.01 0 471011 0 22486 57610 2.562038601797 574358.0 507059.0 10338.0 36048.0 806.0 426.0 0.0 66067.0 471011.0 88.3 1.8 6.3 0.1 0.1 0.0 11.5 82.0 50 50 50.00 28 28717900 26.6 22.6 22.6 28.3 0.0 36.6 23.8 smartseq 1484085 SRR4251529 SRP090061 SRS1699205 SRX2171530 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317748: 26Dp3_A03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317748 GSM2317748 26Dp3_A03_smart-seq 169746200 1697462 2016-09-30 15:56:31 119285085 169746200 1697462 2 1697462 index:0,count:1697462,average:50,stdev:0|index:1,count:1697462,average:50,stdev:0 GSM2317748_r1 2.64 3.32 0.15 144816928 160214523 137484641 154011914 110.63 112.02 1561226 1470765 195.177 936.371 100 7567 40.77 43.01 1849583 636508 1849583 636508 41.12 41.4 1849583 641924 1849583 612755 49731578 34.34 2.19 0 4.79 0 0.14 0 0.33 0 0.00 0 7.56 0 1561226 0 100 0 98.69 0 1.55 0 0.03 0 1.19 0 0.01 0 265.69 0 0.26 0 37122 0 1697462 0 81298 0 2373 0 5546 0 0 0 128317 0 53 0 0 0 566 0 56430 0 3450 0 60499 0 87.18 0 1479928 0 10220 58308 5.705283757339 1697462.0 1561226.0 37122.0 81298.0 2373.0 5546.0 0.0 128317.0 1479928.0 92.0 2.2 4.8 0.1 0.3 0.0 7.6 87.2 50 50 50.00 38 84873100 28.7 20.5 20.6 30.2 0.0 37.3 24.2 smartseq 1484305 SRR4250530 SRP090061 SRS1698207 SRX2170531 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316749: 54Dp1_E01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316749 GSM2316749 54Dp1_E01_smart-seq 163112300 1631123 2016-09-30 15:56:31 76541922 163112300 1631123 2 1631123 index:0,count:1631123,average:50,stdev:0|index:1,count:1631123,average:50,stdev:0 GSM2316749_r1 6.72 2.89 0.17 142640348 191156956 132235864 180763092 134.01 136.7 1495150 1161443 293.297 3067.958 78 3551 78.98 85.41 1865470 1180893 1865470 1180893 79.52 81.58 1865470 1188928 1865470 1127952 10867156 7.62 1.90 0 6.90 0 0.08 0 0.05 0 0.00 0 8.20 0 1495150 0 100 0 98.60 0 1.23 0 0.01 0 1.16 0 0.01 0 234.88 0 0.23 0 30942 0 1631123 0 112589 0 1343 0 808 0 0 0 133822 0 112 0 0 0 1422 0 170834 0 1002 0 173370 0 84.76 0 1382561 0 40013 178780 4.468047884438 1631123.0 1495150.0 30942.0 112589.0 1343.0 808.0 0.0 133822.0 1382561.0 91.7 1.9 6.9 0.1 0.0 0.0 8.2 84.8 50 50 50.00 28 81556150 26.4 22.7 22.9 28.0 0.0 36.2 23.3 smartseq 1484306 SRR4251530 SRP090061 SRS1699207 SRX2171531 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317749: 26Dp3_A04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317749 GSM2317749 26Dp3_A04_smart-seq 221244600 2212446 2016-09-30 15:56:31 154378536 221244600 2212446 2 2212446 index:0,count:2212446,average:50,stdev:0|index:1,count:2212446,average:50,stdev:0 GSM2317749_r1 1.91 3.18 0.14 192915394 220575180 185363117 213184682 114.34 115.01 2076381 1930994 188.900 1117.818 100 10697 45.41 47.31 2298883 942825 2298883 942825 45.71 45.79 2298883 949198 2298883 912565 61521640 31.89 1.60 0 3.77 0 0.19 0 0.37 0 0.00 0 5.59 0 2076381 0 100 0 98.85 0 1.56 0 0.03 0 1.19 0 0.01 0 306.34 0 0.24 0 35463 0 2212446 0 83408 0 4269 0 8169 0 0 0 123627 0 75 0 0 0 765 0 88602 0 2547 0 91989 0 90.08 0 1992973 0 15900 92873 5.841069182390 2212446.0 2076381.0 35463.0 83408.0 4269.0 8169.0 0.0 123627.0 1992973.0 93.9 1.6 3.8 0.2 0.4 0.0 5.6 90.1 50 50 50.00 38 110622300 29.4 19.9 20.0 30.6 0.0 37.4 25.0 smartseq 1484337 SRR4250531 SRP090061 SRS1698208 SRX2170532 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316750: 54Dp1_E02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316750 GSM2316750 54Dp1_E02_smart-seq 118893600 1188936 2016-09-30 15:56:31 55286591 118893600 1188936 2 1188936 index:0,count:1188936,average:50,stdev:0|index:1,count:1188936,average:50,stdev:0 GSM2316750_r1 12.29 2.75 0.07 99234841 137702375 90069895 128183207 138.76 142.32 1051152 840353 273.323 2529.076 81 3072 80.19 88.67 1353716 842893 1353716 842893 82.82 84.99 1353716 870552 1353716 807923 5748187 5.79 2.22 0 8.46 0 0.10 0 0.04 0 0.00 0 11.45 0 1051152 0 100 0 98.50 0 1.15 0 0.01 0 1.13 0 0.01 0 251.77 0 0.23 0 26387 0 1188936 0 100567 0 1208 0 498 0 0 0 136078 0 71 0 0 0 827 0 117450 0 646 0 118994 0 79.95 0 950585 0 27293 121138 4.438427435606 1188936.0 1051152.0 26387.0 100567.0 1208.0 498.0 0.0 136078.0 950585.0 88.4 2.2 8.5 0.1 0.0 0.0 11.4 80.0 50 50 50.00 28 59446800 26.1 22.9 23.0 28.0 0.0 36.2 23.2 smartseq 1484338 SRR4251531 SRP090061 SRS1699209 SRX2171532 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317750: 26Dp3_A05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317750 GSM2317750 26Dp3_A05_smart-seq 172920900 1729209 2016-09-30 15:56:31 120949995 172920900 1729209 2 1729209 index:0,count:1729209,average:50,stdev:0|index:1,count:1729209,average:50,stdev:0 GSM2317750_r1 3.06 3.27 0.16 151402521 176597142 145085341 170340270 116.64 117.41 1622928 1475684 198.826 1233.395 100 7739 50.41 52.67 1813679 818136 1813679 818136 50.79 50.94 1813679 824294 1813679 791362 43782578 28.92 1.61 0 4.02 0 0.09 0 0.19 0 0.00 0 5.86 0 1622928 0 100 0 98.83 0 1.49 0 0.03 0 1.20 0 0.01 0 183.09 0 0.24 0 27756 0 1729209 0 69471 0 1640 0 3328 0 0 0 101313 0 64 0 0 0 791 0 90484 0 2001 0 93340 0 89.84 0 1553457 0 17229 93745 5.441116721806 1729209.0 1622928.0 27756.0 69471.0 1640.0 3328.0 0.0 101313.0 1553457.0 93.9 1.6 4.0 0.1 0.2 0.0 5.9 89.8 50 50 50.00 38 86460450 29.1 20.2 20.4 30.3 0.0 37.3 24.5 smartseq 1484371 SRR4250532 SRP090061 SRS1698209 SRX2170533 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316751: 54Dp1_E03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316751 GSM2316751 54Dp1_E03_smart-seq 124889900 1248899 2016-09-30 15:56:31 59365142 124889900 1248899 2 1248899 index:0,count:1248899,average:50,stdev:0|index:1,count:1248899,average:50,stdev:0 GSM2316751_r1 8.13 3.45 0.09 108199553 148027071 101284855 140610548 136.81 138.83 1140580 887816 284.555 3174.242 81 3151 83.03 88.94 1369735 946991 1369735 946991 83.41 85.36 1369735 951318 1369735 908858 6386831 5.90 1.72 0 6.08 0 0.07 0 0.05 0 0.00 0 8.55 0 1140580 0 100 0 98.69 0 1.21 0 0.01 0 1.13 0 0.01 0 321.15 0 0.22 0 21488 0 1248899 0 75881 0 902 0 594 0 0 0 106823 0 118 0 0 0 949 0 129135 0 650 0 130852 0 85.25 0 1064699 0 37859 132948 3.511661692068 1248899.0 1140580.0 21488.0 75881.0 902.0 594.0 0.0 106823.0 1064699.0 91.3 1.7 6.1 0.1 0.0 0.0 8.6 85.3 50 50 50.00 28 62444950 27.0 22.2 22.4 28.4 0.0 36.1 23.2 smartseq 1484372 SRR4251532 SRP090061 SRS1699206 SRX2171533 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317751: 26Dp3_A06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317751 GSM2317751 26Dp3_A06_smart-seq 179945800 1799458 2016-09-30 15:56:31 125778354 179945800 1799458 2 1799458 index:0,count:1799458,average:50,stdev:0|index:1,count:1799458,average:50,stdev:0 GSM2317751_r1 5.34 2.72 0.14 159480300 186900749 152789395 180087706 117.19 117.87 1703148 1566572 194.652 1137.439 100 8851 49.52 51.75 1887887 843334 1887887 843334 50.14 50.16 1887887 853901 1887887 817452 46403155 29.10 1.51 0 4.08 0 0.10 0 0.22 0 0.00 0 5.03 0 1703148 0 100 0 98.91 0 1.47 0 0.03 0 1.20 0 0.01 0 259.12 0 0.24 0 27155 0 1799458 0 73392 0 1710 0 3999 0 0 0 90601 0 38 0 0 0 657 0 84540 0 2077 0 87312 0 90.57 0 1629756 0 16387 87493 5.339171294319 1799458.0 1703148.0 27155.0 73392.0 1710.0 3999.0 0.0 90601.0 1629756.0 94.6 1.5 4.1 0.1 0.2 0.0 5.0 90.6 50 50 50.00 38 89972900 29.7 19.7 20.0 30.7 0.0 37.4 25.2 smartseq 1484404 SRR4250533 SRP090061 SRS1698210 SRX2170534 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316752: 54Dp1_E04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316752 GSM2316752 54Dp1_E04_smart-seq 187662600 1876626 2016-09-30 15:56:31 87664351 187662600 1876626 2 1876626 index:0,count:1876626,average:50,stdev:0|index:1,count:1876626,average:50,stdev:0 GSM2316752_r1 5.1 3.68 0.08 163213419 212053390 153391803 202317660 129.92 131.9 1723030 1376952 262.984 2816.266 100 5114 75.92 80.97 2046619 1308169 2046619 1308169 76.09 77.6 2046619 1311021 2046619 1253775 17235590 10.56 1.63 0 5.72 0 0.12 0 0.08 0 0.00 0 7.98 0 1723030 0 100 0 98.74 0 1.25 0 0.01 0 1.16 0 0.01 0 281.49 0 0.22 0 30585 0 1876626 0 107329 0 2343 0 1563 0 0 0 149690 0 133 0 0 0 1559 0 187622 0 1008 0 190322 0 86.10 0 1615701 0 40862 196077 4.798516959522 1876626.0 1723030.0 30585.0 107329.0 2343.0 1563.0 0.0 149690.0 1615701.0 91.8 1.6 5.7 0.1 0.1 0.0 8.0 86.1 50 50 50.00 28 93831300 27.1 22.2 22.3 28.4 0.0 36.2 23.9 smartseq 1484405 SRR4251533 SRP090061 SRS1699210 SRX2171534 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317752: 26Dp3_A07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317752 GSM2317752 26Dp3_A07_smart-seq 220296500 2202965 2016-09-30 15:56:31 153659430 220296500 2202965 2 2202965 index:0,count:2202965,average:50,stdev:0|index:1,count:2202965,average:50,stdev:0 GSM2317752_r1 1.99 2.99 0.13 190158433 217841614 182769094 210538804 114.56 115.19 2061746 1909845 180.608 1091.508 100 11614 48.84 50.87 2286764 1006949 2286764 1006949 49.38 49.47 2286764 1018028 2286764 979315 58274279 30.65 1.55 0 3.73 0 0.18 0 0.23 0 0.00 0 6.00 0 2061746 0 100 0 98.81 0 1.47 0 0.03 0 1.18 0 0.01 0 273.47 0 0.23 0 34214 0 2202965 0 82162 0 3985 0 5084 0 0 0 132150 0 66 0 0 0 853 0 101525 0 2318 0 104762 0 89.86 0 1979584 0 14691 106641 7.258934041250 2202965.0 2061746.0 34214.0 82162.0 3985.0 5084.0 0.0 132150.0 1979584.0 93.6 1.6 3.7 0.2 0.2 0.0 6.0 89.9 50 50 50.00 38 110148250 29.3 19.9 20.1 30.7 0.0 37.4 24.8 smartseq 1484433 SRR4250534 SRP090061 SRS1698211 SRX2170535 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316753: 54Dp1_E05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316753 GSM2316753 54Dp1_E05_smart-seq 162747000 1627470 2016-09-30 15:56:31 74497167 162747000 1627470 2 1627470 index:0,count:1627470,average:50,stdev:0|index:1,count:1627470,average:50,stdev:0 GSM2316753_r1 5.34 3.23 0.11 140884912 186797363 132050749 177708408 132.59 134.58 1490080 1191693 257.729 2814.230 100 4566 80.92 86.56 1783413 1205789 1783413 1205789 80.91 82.75 1783413 1205694 1783413 1152786 10009475 7.10 1.69 0 5.96 0 0.10 0 0.04 0 0.00 0 8.30 0 1490080 0 100 0 98.73 0 1.27 0 0.01 0 1.15 0 0.01 0 266.31 0 0.20 0 27565 0 1627470 0 97030 0 1582 0 714 0 0 0 135094 0 154 0 0 0 1115 0 164554 0 884 0 166707 0 85.60 0 1393050 0 34701 170236 4.905795222040 1627470.0 1490080.0 27565.0 97030.0 1582.0 714.0 0.0 135094.0 1393050.0 91.6 1.7 6.0 0.1 0.0 0.0 8.3 85.6 50 50 50.00 28 81373500 27.4 21.7 21.8 29.1 0.0 36.2 23.4 smartseq 1484434 SRR4251534 SRP090061 SRS1699211 SRX2171535 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317753: 26Dp3_A08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317753 GSM2317753 26Dp3_A08_smart-seq 80380000 803800 2016-09-30 15:56:31 57117896 80380000 803800 2 803800 index:0,count:803800,average:50,stdev:0|index:1,count:803800,average:50,stdev:0 GSM2317753_r1 3.94 2.83 0.22 70449416 82021462 67565083 79220357 116.43 117.25 743450 674698 231.075 1537.683 119 2696 47.66 49.75 829622 354335 829622 354335 48.06 48.15 829622 357327 829622 342943 22155943 31.45 1.80 0 3.88 0 0.14 0 0.16 0 0.00 0 7.20 0 743450 0 100 0 98.77 0 1.50 0 0.03 0 1.20 0 0.01 0 170.22 0 0.28 0 14489 0 803800 0 31166 0 1158 0 1325 0 0 0 57867 0 45 0 0 0 328 0 36254 0 958 0 37585 0 88.61 0 712284 0 13423 37125 2.765775162035 803800.0 743450.0 14489.0 31166.0 1158.0 1325.0 0.0 57867.0 712284.0 92.5 1.8 3.9 0.1 0.2 0.0 7.2 88.6 50 50 50.00 38 40190000 28.7 20.5 20.7 30.1 0.0 37.2 23.9 smartseq 1484465 SRR4250535 SRP090061 SRS1698214 SRX2170536 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316754: 54Dp1_E06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316754 GSM2316754 54Dp1_E06_smart-seq 123450900 1234509 2016-09-30 15:56:31 55977332 123450900 1234509 2 1234509 index:0,count:1234509,average:50,stdev:0|index:1,count:1234509,average:50,stdev:0 GSM2316754_r1 8.24 3.32 0.09 104934387 140364420 98431779 133576048 133.76 135.7 1117675 914211 242.702 2499.212 100 3874 77.93 83.32 1334836 870976 1334836 870976 78.24 79.83 1334836 874480 1334836 834443 9306781 8.87 1.74 0 5.86 0 0.09 0 0.07 0 0.00 0 9.30 0 1117675 0 100 0 98.69 0 1.25 0 0.01 0 1.15 0 0.01 0 277.76 0 0.20 0 21493 0 1234509 0 72348 0 1122 0 902 0 0 0 114810 0 77 0 0 0 884 0 120082 0 769 0 121812 0 84.68 0 1045327 0 33960 122373 3.603445229682 1234509.0 1117675.0 21493.0 72348.0 1122.0 902.0 0.0 114810.0 1045327.0 90.5 1.7 5.9 0.1 0.1 0.0 9.3 84.7 50 50 50.00 28 61725450 27.3 21.9 22.1 28.7 0.0 36.3 23.9 smartseq 1484466 SRR4251535 SRP090061 SRS1699212 SRX2171536 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317754: 26Dp3_A09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317754 GSM2317754 26Dp3_A09_smart-seq 92243100 922431 2016-09-30 15:56:31 65338593 92243100 922431 2 922431 index:0,count:922431,average:50,stdev:0|index:1,count:922431,average:50,stdev:0 GSM2317754_r1 4.85 3.07 0.11 79199876 94556369 75059539 90386241 119.39 120.42 843092 749235 225.006 1589.208 110 3224 54.2 57.28 968171 456920 968171 456920 55.0 55.24 968171 463670 968171 440631 18983517 23.97 1.89 0 4.92 0 0.12 0 0.17 0 0.00 0 8.31 0 843092 0 100 0 98.66 0 1.46 0 0.03 0 1.17 0 0.01 0 221.38 0 0.27 0 17433 0 922431 0 45362 0 1122 0 1596 0 0 0 76621 0 37 0 0 0 519 0 54773 0 1217 0 56546 0 86.48 0 797730 0 15281 56456 3.694522609777 922431.0 843092.0 17433.0 45362.0 1122.0 1596.0 0.0 76621.0 797730.0 91.4 1.9 4.9 0.1 0.2 0.0 8.3 86.5 50 50 50.00 38 46121550 27.8 21.2 21.3 29.7 0.0 37.1 23.1 smartseq 1484496 SRR4250536 SRP090061 SRS1698212 SRX2170537 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316755: 54Dp1_E07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316755 GSM2316755 54Dp1_E07_smart-seq 184591200 1845912 2016-09-30 15:56:31 83550693 184591200 1845912 2 1845912 index:0,count:1845912,average:50,stdev:0|index:1,count:1845912,average:50,stdev:0 GSM2316755_r1 7.63 2.87 0.17 160475268 218564612 151070358 208044309 136.2 137.71 1710556 1395145 240.090 2377.497 100 6180 81.56 86.87 1996371 1395137 1996371 1395137 81.6 83.09 1996371 1395812 1996371 1334498 11144741 6.94 1.70 0 5.66 0 0.06 0 0.05 0 0.00 0 7.23 0 1710556 0 100 0 98.73 0 1.22 0 0.01 0 1.15 0 0.01 0 265.81 0 0.20 0 31377 0 1845912 0 104517 0 1038 0 924 0 0 0 133394 0 141 0 0 0 1356 0 191004 0 934 0 193435 0 87.01 0 1606039 0 39343 197192 5.012124138983 1845912.0 1710556.0 31377.0 104517.0 1038.0 924.0 0.0 133394.0 1606039.0 92.7 1.7 5.7 0.1 0.1 0.0 7.2 87.0 50 50 50.00 28 92295600 27.6 21.6 21.7 29.0 0.0 36.3 24.0 smartseq 1484497 SRR4251536 SRP090061 SRS1699213 SRX2171537 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317755: 26Dp3_A10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317755 GSM2317755 26Dp3_A10_smart-seq 123010000 1230100 2016-09-30 15:56:31 87794145 123010000 1230100 2 1230100 index:0,count:1230100,average:50,stdev:0|index:1,count:1230100,average:50,stdev:0 GSM2317755_r1 9.47 2.88 0.12 105796960 133108182 100061529 127073098 125.81 126.99 1121762 997398 224.546 1580.715 105 4386 59.7 63.24 1294783 669673 1294783 669673 60.73 61.04 1294783 681288 1294783 646419 22170228 20.96 1.77 0 5.11 0 0.11 0 0.17 0 0.00 0 8.52 0 1121762 0 100 0 98.67 0 1.46 0 0.02 0 1.18 0 0.01 0 276.77 0 0.28 0 21804 0 1230100 0 62798 0 1385 0 2099 0 0 0 104854 0 58 0 0 0 748 0 75405 0 1415 0 77626 0 86.09 0 1058964 0 16392 78117 4.765556368960 1230100.0 1121762.0 21804.0 62798.0 1385.0 2099.0 0.0 104854.0 1058964.0 91.2 1.8 5.1 0.1 0.2 0.0 8.5 86.1 50 50 50.00 38 61505000 27.4 21.7 21.8 29.1 0.0 37.0 23.2 smartseq 1484529 SRR4250537 SRP090061 SRS1698213 SRX2170538 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316756: 54Dp1_E08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316756 GSM2316756 54Dp1_E08_smart-seq 239227800 2392278 2016-09-30 15:56:31 106166438 239227800 2392278 2 2392278 index:0,count:2392278,average:50,stdev:0|index:1,count:2392278,average:50,stdev:0 GSM2316756_r1 6.02 3.17 0.23 203161737 268991290 190297744 255582524 132.4 134.31 2199893 1859339 204.467 2005.513 100 9955 78.9 84.48 2620597 1735606 2620597 1735606 79.46 81.2 2620597 1747951 2620597 1668103 15988148 7.87 1.75 0 6.08 0 0.13 0 0.10 0 0.00 0 7.82 0 2199893 0 100 0 98.70 0 1.29 0 0.01 0 1.14 0 0.01 0 318.97 0 0.20 0 41813 0 2392278 0 145523 0 3053 0 2315 0 0 0 187017 0 162 0 0 0 1557 0 223730 0 1276 0 226725 0 85.88 0 2054370 0 36027 229919 6.381852499514 2392278.0 2199893.0 41813.0 145523.0 3053.0 2315.0 0.0 187017.0 2054370.0 92.0 1.7 6.1 0.1 0.1 0.0 7.8 85.9 50 50 50.00 28 119613900 28.3 21.0 21.1 29.6 0.0 36.4 24.4 smartseq 1484530 SRR4251537 SRP090061 SRS1699219 SRX2171538 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317756: 26Dp3_A11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317756 GSM2317756 26Dp3_A11_smart-seq 154274500 1542745 2016-09-30 15:56:31 109697961 154274500 1542745 2 1542745 index:0,count:1542745,average:50,stdev:0|index:1,count:1542745,average:50,stdev:0 GSM2317756_r1 2.91 2.82 0.11 135388247 156610809 129664012 150680580 115.68 116.21 1427990 1300955 232.223 1488.279 100 5312 46.41 48.51 1595091 662702 1595091 662702 46.61 46.57 1595091 665526 1595091 636187 43125179 31.85 1.76 0 4.02 0 0.13 0 0.20 0 0.00 0 7.10 0 1427990 0 100 0 98.85 0 1.49 0 0.03 0 1.19 0 0.01 0 292.31 0 0.27 0 27131 0 1542745 0 61952 0 2028 0 3145 0 0 0 109582 0 56 0 0 0 798 0 72307 0 1980 0 75141 0 88.55 0 1366038 0 15400 75884 4.927532467532 1542745.0 1427990.0 27131.0 61952.0 2028.0 3145.0 0.0 109582.0 1366038.0 92.6 1.8 4.0 0.1 0.2 0.0 7.1 88.5 50 50 50.00 38 77137250 27.8 21.3 21.5 29.4 0.0 37.1 23.7 smartseq 1484562 SRR4250538 SRP090061 SRS1698215 SRX2170539 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316757: 54Dp1_E09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316757 GSM2316757 54Dp1_E09_smart-seq 153590900 1535909 2016-09-30 15:56:31 67775244 153590900 1535909 2 1535909 index:0,count:1535909,average:50,stdev:0|index:1,count:1535909,average:50,stdev:0 GSM2316757_r1 6.45 3.51 0.21 131096016 173161770 122420305 164262482 132.09 134.18 1415281 1180613 210.545 2258.515 79 6099 79.52 85.41 1699712 1125375 1699712 1125375 80.01 81.8 1699712 1132394 1699712 1077789 9624538 7.34 1.80 0 6.36 0 0.10 0 0.09 0 0.00 0 7.66 0 1415281 0 100 0 98.66 0 1.23 0 0.01 0 1.15 0 0.01 0 190.66 0 0.19 0 27721 0 1535909 0 97714 0 1493 0 1433 0 0 0 117702 0 120 0 0 0 1026 0 154454 0 834 0 156434 0 85.78 0 1317567 0 33307 158848 4.769207674063 1535909.0 1415281.0 27721.0 97714.0 1493.0 1433.0 0.0 117702.0 1317567.0 92.1 1.8 6.4 0.1 0.1 0.0 7.7 85.8 50 50 50.00 28 76795450 27.9 21.4 21.4 29.3 0.0 36.4 24.4 smartseq 1484563 SRR4251538 SRP090061 SRS1699214 SRX2171539 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317757: 26Dp3_B01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317757 GSM2317757 26Dp3_B01_smart-seq 91121600 911216 2016-09-30 15:56:31 63808208 91121600 911216 2 911216 index:0,count:911216,average:50,stdev:0|index:1,count:911216,average:50,stdev:0 GSM2317757_r1 3.74 2.83 0.29 78029173 85568084 73881080 82357339 109.66 111.47 825999 770127 235.605 1269.577 132 3024 41.66 44.07 995158 344140 995158 344140 42.17 42.69 995158 348340 995158 333364 23943412 30.69 1.67 0 4.96 0 0.17 0 0.23 0 0.00 0 8.95 0 825999 0 100 0 98.72 0 1.48 0 0.02 0 1.22 0 0.01 0 252.34 0 0.26 0 15262 0 911216 0 45178 0 1565 0 2056 0 0 0 81596 0 46 0 0 0 366 0 28378 0 796 0 29586 0 85.69 0 780821 0 9023 29916 3.315526986590 911216.0 825999.0 15262.0 45178.0 1565.0 2056.0 0.0 81596.0 780821.0 90.6 1.7 5.0 0.2 0.2 0.0 9.0 85.7 50 50 50.00 38 45560800 28.6 20.2 20.2 31.0 0.0 37.1 22.6 smartseq 1484592 SRR4250539 SRP090061 SRS1698216 SRX2170540 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316758: 54Dp1_E10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316758 GSM2316758 54Dp1_E10_smart-seq 136053700 1360537 2016-09-30 15:56:31 62376266 136053700 1360537 2 1360537 index:0,count:1360537,average:50,stdev:0|index:1,count:1360537,average:50,stdev:0 GSM2316758_r1 6.43 2.99 0.16 117557946 156199332 109543504 147798259 132.87 134.92 1243114 995148 248.573 2782.791 100 4130 79.1 85.11 1511034 983353 1511034 983353 79.68 81.5 1511034 990508 1511034 941539 9725345 8.27 1.64 0 6.45 0 0.08 0 0.06 0 0.00 0 8.50 0 1243114 0 100 0 98.72 0 1.25 0 0.01 0 1.14 0 0.01 0 288.11 0 0.21 0 22344 0 1360537 0 87791 0 1039 0 788 0 0 0 115596 0 98 0 0 0 1109 0 142391 0 802 0 144400 0 84.92 0 1155323 0 34575 147628 4.269790310918 1360537.0 1243114.0 22344.0 87791.0 1039.0 788.0 0.0 115596.0 1155323.0 91.4 1.6 6.5 0.1 0.1 0.0 8.5 84.9 50 50 50.00 28 68026850 26.9 22.2 22.4 28.5 0.0 36.3 23.5 smartseq 1484593 SRR4251539 SRP090061 SRS1699216 SRX2171540 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317758: 26Dp3_B02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317758 GSM2317758 26Dp3_B02_smart-seq 246502800 2465028 2016-09-30 15:56:31 168792399 246502800 2465028 2 2465028 index:0,count:2465028,average:50,stdev:0|index:1,count:2465028,average:50,stdev:0 GSM2317758_r1 2.32 2.9 0.18 215090324 238747722 208134800 232749549 111.0 111.83 2321962 2224137 177.611 841.972 100 13720 36.92 38.18 2524282 857190 2524282 857190 37.03 37.2 2524282 859724 2524282 835142 82366919 38.29 1.42 0 3.13 0 0.15 0 0.26 0 0.00 0 5.40 0 2321962 0 100 0 98.99 0 1.51 0 0.03 0 1.22 0 0.01 0 385.83 0 0.21 0 35094 0 2465028 0 77062 0 3683 0 6324 0 0 0 133059 0 73 0 0 0 686 0 60131 0 2267 0 63157 0 91.07 0 2244900 0 9759 62625 6.417153396864 2465028.0 2321962.0 35094.0 77062.0 3683.0 6324.0 0.0 133059.0 2244900.0 94.2 1.4 3.1 0.1 0.3 0.0 5.4 91.1 50 50 50.00 38 123251400 30.0 19.3 19.4 31.4 0.0 37.5 24.7 smartseq 1484816 SRR4250540 SRP090061 SRS1698217 SRX2170541 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316759: 54Dp1_E11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316759 GSM2316759 54Dp1_E11_smart-seq 119055300 1190553 2016-09-30 15:56:31 56458582 119055300 1190553 2 1190553 index:0,count:1190553,average:50,stdev:0|index:1,count:1190553,average:50,stdev:0 GSM2316759_r1 7.14 3.07 0.07 103684111 141036223 95898807 132955744 136.02 138.64 1086804 842390 292.093 3030.391 91 2731 80.7 87.48 1354626 877067 1354626 877067 81.64 83.8 1354626 887256 1354626 840197 6708148 6.47 1.75 0 7.07 0 0.08 0 0.07 0 0.00 0 8.56 0 1086804 0 100 0 98.67 0 1.23 0 0.01 0 1.15 0 0.01 0 285.73 0 0.23 0 20865 0 1190553 0 84203 0 987 0 846 0 0 0 101916 0 87 0 0 0 957 0 124358 0 685 0 126087 0 84.21 0 1002601 0 32739 128632 3.929014325422 1190553.0 1086804.0 20865.0 84203.0 987.0 846.0 0.0 101916.0 1002601.0 91.3 1.8 7.1 0.1 0.1 0.0 8.6 84.2 50 50 50.00 28 59527650 26.5 22.6 22.8 28.1 0.0 36.1 22.9 smartseq 1484817 SRR4251540 SRP090061 SRS1699215 SRX2171541 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317759: 26Dp3_B03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317759 GSM2317759 26Dp3_B03_smart-seq 312613600 3126136 2016-09-30 15:56:31 213413040 312613600 3126136 2 3126136 index:0,count:3126136,average:50,stdev:0|index:1,count:3126136,average:50,stdev:0 GSM2317759_r1 4.76 3.16 0.46 274441162 313759468 263895137 303680214 114.33 115.08 2982267 2830680 174.817 820.344 100 16975 40.82 42.51 3279419 1217449 3279419 1217449 41.24 41.39 3279419 1229892 3279419 1185357 96769522 35.26 1.53 0 3.79 0 0.19 0 0.21 0 0.00 0 4.20 0 2982267 0 100 0 98.92 0 1.51 0 0.03 0 1.18 0 0.01 0 234.46 0 0.21 0 47740 0 3126136 0 118394 0 5835 0 6693 0 0 0 131341 0 69 0 0 0 779 0 95936 0 2958 0 99742 0 91.61 0 2863873 0 17057 101047 5.924078091106 3126136.0 2982267.0 47740.0 118394.0 5835.0 6693.0 0.0 131341.0 2863873.0 95.4 1.5 3.8 0.2 0.2 0.0 4.2 91.6 50 50 50.00 38 156306800 30.5 19.0 19.1 31.3 0.0 37.7 26.4 smartseq 1484849 SRR4250541 SRP090061 SRS1698219 SRX2170542 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316760: 54Dp1_F01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316760 GSM2316760 54Dp1_F01_smart-seq 201444700 2014447 2016-09-30 15:56:31 90515947 201444700 2014447 2 2014447 index:0,count:2014447,average:50,stdev:0|index:1,count:2014447,average:50,stdev:0 GSM2316760_r1 6.41 3.16 0.11 172146444 228838084 160966552 217890554 132.93 135.36 1828829 1478284 256.200 2681.410 79 5764 76.27 81.8 2245029 1394846 2245029 1394846 76.3 78.11 2245029 1395446 2245029 1332010 16862815 9.80 1.90 0 6.13 0 0.09 0 0.06 0 0.00 0 9.06 0 1828829 0 100 0 98.56 0 1.26 0 0.01 0 1.15 0 0.01 0 302.17 0 0.21 0 38345 0 2014447 0 123556 0 1887 0 1142 0 0 0 182589 0 122 0 0 0 1683 0 205277 0 1276 0 208358 0 84.65 0 1705273 0 40097 213355 5.320971643764 2014447.0 1828829.0 38345.0 123556.0 1887.0 1142.0 0.0 182589.0 1705273.0 90.8 1.9 6.1 0.1 0.1 0.0 9.1 84.7 50 50 50.00 28 100722350 26.4 22.7 22.8 28.2 0.0 36.5 23.7 smartseq 1484850 SRR4251541 SRP090061 SRS1699218 SRX2171542 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317760: 26Dp3_B04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317760 GSM2317760 26Dp3_B04_smart-seq 359424000 3594240 2016-09-30 15:56:31 243614745 359424000 3594240 2 3594240 index:0,count:3594240,average:50,stdev:0|index:1,count:3594240,average:50,stdev:0 GSM2317760_r1 3.68 3.55 0.23 309279889 355764447 296783422 343653636 115.03 115.79 3421775 3231716 155.269 768.334 100 22482 45.57 47.57 3782381 1559273 3782381 1559273 46.13 46.19 3782381 1578405 3782381 1514077 97946942 31.67 1.56 0 4.00 0 0.17 0 0.26 0 0.00 0 4.37 0 3421775 0 100 0 98.83 0 1.49 0 0.03 0 1.21 0 0.01 0 369.69 0 0.20 0 56054 0 3594240 0 143700 0 6086 0 9185 0 0 0 157194 0 118 0 0 0 1270 0 138952 0 3129 0 143469 0 91.20 0 3278075 0 15127 141079 9.326303959807 3594240.0 3421775.0 56054.0 143700.0 6086.0 9185.0 0.0 157194.0 3278075.0 95.2 1.6 4.0 0.2 0.3 0.0 4.4 91.2 50 50 50.00 38 179712000 30.7 18.8 18.9 31.6 0.0 37.7 26.4 smartseq 1484881 SRR4250542 SRP090061 SRS1698218 SRX2170543 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316761: 54Dp1_F02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316761 GSM2316761 54Dp1_F02_smart-seq 260086200 2600862 2016-09-30 15:56:31 115911639 260086200 2600862 2 2600862 index:0,count:2600862,average:50,stdev:0|index:1,count:2600862,average:50,stdev:0 GSM2316761_r1 7.99 3.07 0.17 219563547 296301105 200098651 276838954 134.95 138.35 2328923 1909993 250.824 2445.388 100 7419 77.8 85.65 3046373 1811927 3046373 1811927 79.15 81.81 3046373 1843228 3046373 1730601 16446816 7.49 1.87 0 8.21 0 0.09 0 0.05 0 0.00 0 10.32 0 2328923 0 100 0 98.59 0 1.20 0 0.01 0 1.15 0 0.01 0 360.12 0 0.21 0 48751 0 2600862 0 213432 0 2294 0 1297 0 0 0 268348 0 154 0 0 0 2043 0 249217 0 1411 0 252825 0 81.34 0 2115491 0 29762 260614 8.756602378872 2600862.0 2328923.0 48751.0 213432.0 2294.0 1297.0 0.0 268348.0 2115491.0 89.5 1.9 8.2 0.1 0.0 0.0 10.3 81.3 50 50 50.00 28 130043100 26.3 22.9 22.9 27.9 0.0 36.5 23.7 smartseq 1484882 SRR4251542 SRP090061 SRS1699217 SRX2171543 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317761: 26Dp3_B05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317761 GSM2317761 26Dp3_B05_smart-seq 331548800 3315488 2016-09-30 15:56:31 225226995 331548800 3315488 2 3315488 index:0,count:3315488,average:50,stdev:0|index:1,count:3315488,average:50,stdev:0 GSM2317761_r1 3.27 3.77 0.25 289935881 330750493 280052466 320774185 114.08 114.54 3166624 2984544 167.280 868.610 100 19023 42.96 44.53 3434262 1360295 3434262 1360295 43.28 43.22 3434262 1370613 3434262 1320227 99650459 34.37 1.58 0 3.37 0 0.12 0 0.29 0 0.00 0 4.08 0 3166624 0 100 0 98.91 0 1.49 0 0.03 0 1.20 0 0.01 0 385.02 0 0.20 0 52376 0 3315488 0 111628 0 4094 0 9611 0 0 0 135159 0 99 0 0 0 1146 0 124258 0 3300 0 128803 0 92.14 0 3054996 0 17798 128293 7.208281829419 3315488.0 3166624.0 52376.0 111628.0 4094.0 9611.0 0.0 135159.0 3054996.0 95.5 1.6 3.4 0.1 0.3 0.0 4.1 92.1 50 50 50.00 38 165774400 30.5 19.1 19.2 31.3 0.0 37.6 26.4 smartseq 1484915 SRR4250543 SRP090061 SRS1698220 SRX2170544 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316762: 54Dp1_F03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316762 GSM2316762 54Dp1_F03_smart-seq 380587200 3805872 2016-09-30 15:56:31 166805471 380587200 3805872 2 3805872 index:0,count:3805872,average:50,stdev:0|index:1,count:3805872,average:50,stdev:0 GSM2316762_r1 3.7 2.92 0.09 328484574 428070448 305432540 405646750 130.32 132.81 3485449 2891153 227.437 2226.304 100 13216 76.52 82.49 4317527 2666931 4317527 2666931 76.68 78.68 4317527 2672512 4317527 2543936 32353654 9.85 1.68 0 6.63 0 0.08 0 0.08 0 0.00 0 8.26 0 3485449 0 100 0 98.71 0 1.32 0 0.01 0 1.16 0 0.01 0 334.17 0 0.20 0 64027 0 3805872 0 252353 0 3009 0 3051 0 0 0 314363 0 359 0 0 0 2961 0 383548 0 2147 0 389015 0 84.95 0 3233096 0 41041 398434 9.708194244780 3805872.0 3485449.0 64027.0 252353.0 3009.0 3051.0 0.0 314363.0 3233096.0 91.6 1.7 6.6 0.1 0.1 0.0 8.3 85.0 50 50 50.00 28 190293600 27.2 22.0 22.1 28.7 0.0 36.6 24.1 smartseq 1484916 SRR4251543 SRP090061 SRS1699220 SRX2171544 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317762: 26Dp3_B06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317762 GSM2317762 26Dp3_B06_smart-seq 408055300 4080553 2016-09-30 15:56:31 277185317 408055300 4080553 2 4080553 index:0,count:4080553,average:50,stdev:0|index:1,count:4080553,average:50,stdev:0 GSM2317762_r1 3.97 3.12 0.27 351405233 409874281 336732088 394308303 116.64 117.1 3898756 3656222 153.728 820.005 100 26851 50.7 53.0 4289148 1976632 4289148 1976632 51.41 51.44 4289148 2004427 4289148 1918625 99686167 28.37 1.60 0 4.15 0 0.09 0 0.18 0 0.00 0 4.18 0 3898756 0 100 0 98.81 0 1.52 0 0.03 0 1.16 0 0.01 0 408.06 0 0.20 0 65183 0 4080553 0 169212 0 3562 0 7501 0 0 0 170734 0 231 0 0 0 1325 0 187036 0 3522 0 192114 0 91.40 0 3729544 0 19420 195603 10.072245108136 4080553.0 3898756.0 65183.0 169212.0 3562.0 7501.0 0.0 170734.0 3729544.0 95.5 1.6 4.1 0.1 0.2 0.0 4.2 91.4 50 50 50.00 38 204027650 30.5 19.0 19.2 31.3 0.0 37.6 26.7 smartseq 1484948 SRR4250544 SRP090061 SRS1698221 SRX2170545 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316763: 54Dp1_F04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316763 GSM2316763 54Dp1_F04_smart-seq 286867700 2868677 2016-09-30 15:56:31 123439900 286867700 2868677 2 2868677 index:0,count:2868677,average:50,stdev:0|index:1,count:2868677,average:50,stdev:0 GSM2316763_r1 10.79 2.66 0.12 240234263 327303095 224949436 310941481 136.24 138.23 2610962 2238034 201.610 1913.082 69 12601 79.17 84.86 3118774 2067025 3118774 2067025 80.02 81.74 3118774 2089323 3118774 1991094 20045563 8.34 1.75 0 6.10 0 0.11 0 0.08 0 0.00 0 8.79 0 2610962 0 100 0 98.66 0 1.21 0 0.01 0 1.14 0 0.01 0 322.73 0 0.19 0 50067 0 2868677 0 175016 0 3265 0 2381 0 0 0 252069 0 150 0 0 0 1749 0 258450 0 1364 0 261713 0 84.92 0 2435946 0 36686 266834 7.273455814207 2868677.0 2610962.0 50067.0 175016.0 3265.0 2381.0 0.0 252069.0 2435946.0 91.0 1.7 6.1 0.1 0.1 0.0 8.8 84.9 50 50 50.00 28 143433850 27.9 21.4 21.4 29.3 0.0 36.6 24.7 smartseq 1484949 SRR4251544 SRP090061 SRS1699221 SRX2171545 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317763: 26Dp3_B07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317763 GSM2317763 26Dp3_B07_smart-seq 441483300 4414833 2016-09-30 15:56:31 300523334 441483300 4414833 2 4414833 index:0,count:4414833,average:50,stdev:0|index:1,count:4414833,average:50,stdev:0 GSM2317763_r1 4.04 3.01 0.21 378050370 436611126 364133827 422763567 115.49 116.1 4215235 3968778 154.395 846.647 71 27572 44.46 46.23 4610549 1874114 4610549 1874114 44.85 44.86 4610549 1890681 4610549 1818672 126728580 33.52 1.67 0 3.66 0 0.13 0 0.26 0 0.00 0 4.13 0 4215235 0 100 0 98.80 0 1.50 0 0.03 0 1.19 0 0.01 0 378.41 0 0.20 0 73911 0 4414833 0 161406 0 5856 0 11570 0 0 0 182172 0 220 0 0 0 1549 0 183797 0 4102 0 189668 0 91.82 0 4053829 0 25661 189324 7.377888624761 4414833.0 4215235.0 73911.0 161406.0 5856.0 11570.0 0.0 182172.0 4053829.0 95.5 1.7 3.7 0.1 0.3 0.0 4.1 91.8 50 50 50.00 38 220741650 30.6 19.0 19.1 31.4 0.0 37.6 26.9 smartseq 1484981 SRR4250545 SRP090061 SRS1698222 SRX2170546 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316764: 54Dp1_F05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316764 GSM2316764 54Dp1_F05_smart-seq 237697600 2376976 2016-09-30 15:56:31 103341106 237697600 2376976 2 2376976 index:0,count:2376976,average:50,stdev:0|index:1,count:2376976,average:50,stdev:0 GSM2316764_r1 7.11 2.89 0.08 199431676 272915215 180870345 253390787 136.85 140.1 2166702 1769979 219.326 1743.621 69 9617 79.8 88.41 2776311 1729114 2776311 1729114 83.15 85.21 2776311 1801566 2776311 1666677 11482745 5.76 2.35 0 8.87 0 0.09 0 0.07 0 0.00 0 8.69 0 2166702 0 100 0 98.44 0 1.25 0 0.01 0 1.15 0 0.01 0 305.61 0 0.20 0 55823 0 2376976 0 210827 0 2185 0 1577 0 0 0 206512 0 99 0 0 0 1633 0 288569 0 1291 0 291592 0 82.28 0 1955875 0 34451 295274 8.570839743404 2376976.0 2166702.0 55823.0 210827.0 2185.0 1577.0 0.0 206512.0 1955875.0 91.2 2.3 8.9 0.1 0.1 0.0 8.7 82.3 50 50 50.00 28 118848800 27.0 22.4 22.4 28.2 0.0 36.6 24.8 smartseq 1484982 SRR4251545 SRP090061 SRS1699227 SRX2171546 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317764: 26Dp3_B08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317764 GSM2317764 26Dp3_B08_smart-seq 171309900 1713099 2016-09-30 15:56:31 120198092 171309900 1713099 2 1713099 index:0,count:1713099,average:50,stdev:0|index:1,count:1713099,average:50,stdev:0 GSM2317764_r1 5.54 3.07 0.17 147056396 178784277 138340115 169822567 121.58 122.76 1598497 1453450 181.847 1270.053 100 8729 54.61 58.12 1870933 872866 1870933 872866 55.72 55.82 1870933 890623 1870933 838358 34687930 23.59 1.88 0 5.64 0 0.14 0 0.23 0 0.00 0 6.33 0 1598497 0 100 0 98.67 0 1.45 0 0.02 0 1.19 0 0.01 0 212.66 0 0.25 0 32208 0 1713099 0 96637 0 2344 0 3897 0 0 0 108361 0 60 0 0 0 1045 0 106044 0 2188 0 109337 0 87.67 0 1501860 0 16117 110634 6.864428863932 1713099.0 1598497.0 32208.0 96637.0 2344.0 3897.0 0.0 108361.0 1501860.0 93.3 1.9 5.6 0.1 0.2 0.0 6.3 87.7 50 50 50.00 38 85654950 27.1 22.3 22.4 28.2 0.0 37.2 24.7 smartseq 1485014 SRR4250546 SRP090061 SRS1698225 SRX2170547 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316765: 54Dp1_F06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316765 GSM2316765 54Dp1_F06_smart-seq 216784800 2167848 2016-09-30 15:56:31 94045601 216784800 2167848 2 2167848 index:0,count:2167848,average:50,stdev:0|index:1,count:2167848,average:50,stdev:0 GSM2316765_r1 6.63 3.06 0.17 182431513 243284491 170482089 230683818 133.36 135.31 1975461 1688276 207.185 1974.712 69 8734 78.79 84.6 2362682 1556457 2362682 1556457 79.36 81.07 2362682 1567687 2362682 1491501 14456757 7.92 1.77 0 6.26 0 0.09 0 0.05 0 0.00 0 8.73 0 1975461 0 100 0 98.70 0 1.33 0 0.01 0 1.16 0 0.01 0 354.74 0 0.19 0 38397 0 2167848 0 135640 0 1972 0 1062 0 0 0 189353 0 181 0 0 0 1516 0 192536 0 1082 0 195315 0 84.87 0 1839821 0 34876 197830 5.672382153917 2167848.0 1975461.0 38397.0 135640.0 1972.0 1062.0 0.0 189353.0 1839821.0 91.1 1.8 6.3 0.1 0.0 0.0 8.7 84.9 50 50 50.00 28 108392400 28.2 21.0 21.0 29.8 0.0 36.6 24.3 smartseq 1485015 SRR4251546 SRP090061 SRS1699226 SRX2171547 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317765: 26Dp3_B09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317765 GSM2317765 26Dp3_B09_smart-seq 201129100 2011291 2016-09-30 15:56:31 137479741 201129100 2011291 2 2011291 index:0,count:2011291,average:50,stdev:0|index:1,count:2011291,average:50,stdev:0 GSM2317765_r1 2.1 3.14 0.26 178983353 202168117 172643006 196065221 112.95 113.57 1919814 1788912 191.525 1062.629 100 9777 40.17 41.69 2093700 771213 2093700 771213 40.49 40.47 2093700 777343 2093700 748698 64149054 35.84 1.49 0 3.47 0 0.14 0 0.21 0 0.00 0 4.20 0 1919814 0 100 0 98.97 0 1.54 0 0.03 0 1.19 0 0.01 0 268.17 0 0.21 0 29958 0 2011291 0 69825 0 2735 0 4195 0 0 0 84547 0 93 0 0 0 707 0 75847 0 2229 0 78876 0 91.98 0 1849989 0 21237 78279 3.685972594999 2011291.0 1919814.0 29958.0 69825.0 2735.0 4195.0 0.0 84547.0 1849989.0 95.5 1.5 3.5 0.1 0.2 0.0 4.2 92.0 50 50 50.00 38 100564550 30.2 19.3 19.4 31.1 0.0 37.6 25.9 smartseq 1485044 SRR4250547 SRP090061 SRS1698224 SRX2170548 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316766: 54Dp1_F07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316766 GSM2316766 54Dp1_F07_smart-seq 211094600 2110946 2016-09-30 15:56:31 91252442 211094600 2110946 2 2110946 index:0,count:2110946,average:50,stdev:0|index:1,count:2110946,average:50,stdev:0 GSM2316766_r1 3.67 3.03 0.16 175693177 227683550 166293177 217817181 129.59 130.98 1905572 1644890 203.827 1870.270 100 8699 75.82 80.33 2201557 1444823 2201557 1444823 75.93 77.28 2201557 1446987 2201557 1389821 18668747 10.63 1.63 0 5.07 0 0.08 0 0.05 0 0.00 0 9.60 0 1905572 0 100 0 98.76 0 1.33 0 0.01 0 1.17 0 0.01 0 253.31 0 0.18 0 34436 0 2110946 0 107045 0 1609 0 1020 0 0 0 202745 0 185 0 0 0 1387 0 174158 0 955 0 176685 0 85.20 0 1798527 0 32792 179463 5.472767748231 2110946.0 1905572.0 34436.0 107045.0 1609.0 1020.0 0.0 202745.0 1798527.0 90.3 1.6 5.1 0.1 0.0 0.0 9.6 85.2 50 50 50.00 28 105547300 28.8 20.4 20.2 30.5 0.0 36.6 24.1 smartseq 1485045 SRR4251547 SRP090061 SRS1699222 SRX2171548 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317766: 26Dp3_B10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317766 GSM2317766 26Dp3_B10_smart-seq 139517800 1395178 2016-09-30 15:56:31 98097223 139517800 1395178 2 1395178 index:0,count:1395178,average:50,stdev:0|index:1,count:1395178,average:50,stdev:0 GSM2317766_r1 2.02 2.79 0.17 123369324 140863008 118578536 136445406 114.18 115.07 1299224 1183255 233.542 1468.750 110 4802 43.49 45.29 1450586 565072 1450586 565072 43.66 43.77 1450586 567281 1450586 546106 41370689 33.53 1.70 0 3.70 0 0.14 0 0.18 0 0.00 0 6.56 0 1299224 0 100 0 98.87 0 1.51 0 0.03 0 1.20 0 0.01 0 313.92 0 0.26 0 23776 0 1395178 0 51612 0 1950 0 2475 0 0 0 91529 0 67 0 0 0 641 0 60101 0 2029 0 62838 0 89.42 0 1247612 0 17770 62723 3.529712999437 1395178.0 1299224.0 23776.0 51612.0 1950.0 2475.0 0.0 91529.0 1247612.0 93.1 1.7 3.7 0.1 0.2 0.0 6.6 89.4 50 50 50.00 38 69758900 28.6 20.5 20.6 30.2 0.0 37.1 23.7 smartseq 1485074 SRR4250548 SRP090061 SRS1698223 SRX2170549 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316767: 54Dp1_F08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316767 GSM2316767 54Dp1_F08_smart-seq 290986000 2909860 2016-09-30 15:56:31 124887092 290986000 2909860 2 2909860 index:0,count:2909860,average:50,stdev:0|index:1,count:2909860,average:50,stdev:0 GSM2316767_r1 4.6 3.19 0.13 234466858 299971224 220512096 284964798 127.94 129.23 2586616 2269543 182.486 1504.431 78 13944 72.94 77.79 3020916 1886672 3020916 1886672 73.51 74.64 3020916 1901495 3020916 1810183 27913792 11.91 1.82 0 5.55 0 0.14 0 0.11 0 0.00 0 10.87 0 2586616 0 100 0 98.63 0 1.32 0 0.01 0 1.16 0 0.01 0 299.30 0 0.18 0 52878 0 2909860 0 161395 0 3979 0 3094 0 0 0 316171 0 176 0 0 0 1547 0 238140 0 1365 0 241228 0 83.34 0 2425221 0 27012 246250 9.116318673182 2909860.0 2586616.0 52878.0 161395.0 3979.0 3094.0 0.0 316171.0 2425221.0 88.9 1.8 5.5 0.1 0.1 0.0 10.9 83.3 50 50 50.00 28 145493000 28.4 20.9 20.7 30.0 0.0 36.6 24.4 smartseq 1485075 SRR4251548 SRP090061 SRS1699225 SRX2171549 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317767: 26Dp3_B11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317767 GSM2317767 26Dp3_B11_smart-seq 157756100 1577561 2016-09-30 15:56:31 110685421 157756100 1577561 2 1577561 index:0,count:1577561,average:50,stdev:0|index:1,count:1577561,average:50,stdev:0 GSM2317767_r1 3.7 2.82 0.11 138348949 160833523 132717611 155456733 116.25 117.13 1457644 1331223 226.507 1492.920 137 5678 47.36 49.43 1632035 690386 1632035 690386 47.49 47.73 1632035 692303 1632035 666686 42615720 30.80 1.68 0 3.87 0 0.18 0 0.21 0 0.00 0 7.21 0 1457644 0 100 0 98.84 0 1.49 0 0.03 0 1.21 0 0.01 0 258.15 0 0.27 0 26430 0 1577561 0 60982 0 2844 0 3355 0 0 0 113718 0 66 0 0 0 760 0 72136 0 1963 0 74925 0 88.53 0 1396662 0 14490 74512 5.142305037957 1577561.0 1457644.0 26430.0 60982.0 2844.0 3355.0 0.0 113718.0 1396662.0 92.4 1.7 3.9 0.2 0.2 0.0 7.2 88.5 50 50 50.00 38 78878050 27.9 21.2 21.3 29.7 0.0 37.1 23.4 smartseq 1485105 SRR4250549 SRP090061 SRS1698226 SRX2170550 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316768: 54Dp1_F09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316768 GSM2316768 54Dp1_F09_smart-seq 267573000 2675730 2016-09-30 15:56:31 114806894 267573000 2675730 2 2675730 index:0,count:2675730,average:50,stdev:0|index:1,count:2675730,average:50,stdev:0 GSM2316768_r1 4.55 3.64 0.26 222330309 288364085 209453659 274447394 129.7 131.03 2438018 2098523 186.396 1823.709 78 12592 77.69 82.72 2840647 1894108 2840647 1894108 78.15 79.58 2840647 1905229 2840647 1822210 20956909 9.43 1.79 0 5.55 0 0.10 0 0.09 0 0.00 0 8.69 0 2438018 0 100 0 98.65 0 1.32 0 0.01 0 1.14 0 0.01 0 283.31 0 0.18 0 47831 0 2675730 0 148370 0 2605 0 2541 0 0 0 232566 0 230 0 0 0 1728 0 247258 0 1208 0 250424 0 85.57 0 2289648 0 33254 252491 7.592800866061 2675730.0 2438018.0 47831.0 148370.0 2605.0 2541.0 0.0 232566.0 2289648.0 91.1 1.8 5.5 0.1 0.1 0.0 8.7 85.6 50 50 50.00 28 133786500 28.5 20.9 20.7 29.9 0.0 36.6 24.7 smartseq 1485106 SRR4251549 SRP090061 SRS1699224 SRX2171550 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317768: 26Dp3_C01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317768 GSM2317768 26Dp3_C01_smart-seq 139956800 1399568 2016-09-30 15:56:31 97517499 139956800 1399568 2 1399568 index:0,count:1399568,average:50,stdev:0|index:1,count:1399568,average:50,stdev:0 GSM2317768_r1 2.04 2.76 0.15 118205849 152080259 112421918 145424078 128.66 129.36 1263044 1095320 207.466 1679.668 116 5606 82.17 86.52 1410287 1037854 1410287 1037854 82.55 83.58 1410287 1042600 1410287 1002495 8589397 7.27 1.52 0 4.54 0 0.04 0 0.04 0 0.00 0 9.67 0 1263044 0 100 0 98.71 0 1.35 0 0.01 0 1.18 0 0.01 0 279.91 0 0.21 0 21319 0 1399568 0 63555 0 586 0 591 0 0 0 135347 0 66 0 0 0 837 0 107203 0 1102 0 109208 0 85.70 0 1199489 0 13100 112632 8.597862595420 1399568.0 1263044.0 21319.0 63555.0 586.0 591.0 0.0 135347.0 1199489.0 90.2 1.5 4.5 0.0 0.0 0.0 9.7 85.7 50 50 50.00 38 69978400 28.0 20.7 20.8 30.5 0.0 37.1 22.2 smartseq 1485331 SRR4250550 SRP090061 SRS1698227 SRX2170551 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316769: 54Dp1_F10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316769 GSM2316769 54Dp1_F10_smart-seq 175405600 1754056 2016-09-30 15:56:31 76217743 175405600 1754056 2 1754056 index:0,count:1754056,average:50,stdev:0|index:1,count:1754056,average:50,stdev:0 GSM2316769_r1 7.44 2.91 0.18 145312067 193302630 136117093 183790049 133.03 135.02 1579345 1331782 203.553 2064.226 69 7336 77.82 83.36 1898820 1229063 1898820 1229063 78.16 79.86 1898820 1234457 1898820 1177542 12625421 8.69 1.77 0 5.98 0 0.10 0 0.07 0 0.00 0 9.80 0 1579345 0 100 0 98.62 0 1.24 0 0.01 0 1.16 0 0.01 0 332.35 0 0.19 0 31132 0 1754056 0 104896 0 1714 0 1184 0 0 0 171813 0 108 0 0 0 1198 0 168694 0 900 0 170900 0 84.06 0 1474449 0 31041 171504 5.525079733256 1754056.0 1579345.0 31132.0 104896.0 1714.0 1184.0 0.0 171813.0 1474449.0 90.0 1.8 6.0 0.1 0.1 0.0 9.8 84.1 50 50 50.00 28 87702800 27.6 21.7 21.6 29.1 0.0 36.6 24.2 smartseq 1485332 SRR4251550 SRP090061 SRS1699223 SRX2171551 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317769: 26Dp3_C02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317769 GSM2317769 26Dp3_C02_smart-seq 147263900 1472639 2016-09-30 15:56:31 100102463 147263900 1472639 2 1472639 index:0,count:1472639,average:50,stdev:0|index:1,count:1472639,average:50,stdev:0 GSM2317769_r1 3.13 2.79 0.18 129371188 141848725 125463630 138186077 109.64 110.14 1400944 1342041 173.439 719.122 100 8164 37.73 38.95 1512911 528526 1512911 528526 37.99 38.0 1512911 532159 1512911 515687 48292216 37.33 1.37 0 2.98 0 0.16 0 0.28 0 0.00 0 4.43 0 1400944 0 100 0 98.99 0 1.48 0 0.02 0 1.22 0 0.01 0 279.03 0 0.21 0 20193 0 1472639 0 43930 0 2341 0 4096 0 0 0 65258 0 28 0 0 0 418 0 37593 0 1319 0 39358 0 92.15 0 1357014 0 9234 39297 4.255685510071 1472639.0 1400944.0 20193.0 43930.0 2341.0 4096.0 0.0 65258.0 1357014.0 95.1 1.4 3.0 0.2 0.3 0.0 4.4 92.1 50 50 50.00 38 73631950 30.9 18.5 18.8 31.9 0.0 37.7 26.2 smartseq 1485363 SRR4250551 SRP090061 SRS1698229 SRX2170552 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316770: 54Dp1_F11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316770 GSM2316770 54Dp1_F11_smart-seq 185410000 1854100 2016-09-30 15:56:31 81736648 185410000 1854100 2 1854100 index:0,count:1854100,average:50,stdev:0|index:1,count:1854100,average:50,stdev:0 GSM2316770_r1 2.22 2.88 0.19 156932886 200536751 148125645 192146736 127.79 129.72 1677381 1380391 232.568 2403.751 90 6256 74.02 78.6 1999675 1241612 1999675 1241612 73.4 74.94 1999675 1231206 1999675 1183885 18894227 12.04 1.63 0 5.27 0 0.12 0 0.12 0 0.00 0 9.29 0 1677381 0 100 0 98.70 0 1.32 0 0.01 0 1.17 0 0.01 0 266.99 0 0.20 0 30238 0 1854100 0 97708 0 2284 0 2248 0 0 0 172187 0 138 0 0 0 1633 0 182309 0 1168 0 185248 0 85.20 0 1579673 0 33830 188553 5.573544191546 1854100.0 1677381.0 30238.0 97708.0 2284.0 2248.0 0.0 172187.0 1579673.0 90.5 1.6 5.3 0.1 0.1 0.0 9.3 85.2 50 50 50.00 28 92705000 27.2 22.1 22.2 28.5 0.0 36.6 24.3 smartseq 1485364 SRR4251551 SRP090061 SRS1699233 SRX2171552 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317770: 26Dp3_C03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317770 GSM2317770 26Dp3_C03_smart-seq 358604700 3586047 2016-09-30 15:56:31 242507099 358604700 3586047 2 3586047 index:0,count:3586047,average:50,stdev:0|index:1,count:3586047,average:50,stdev:0 GSM2317770_r1 2.61 3.08 0.28 306005214 340007707 294502551 329229366 111.11 111.79 3394614 3266823 151.468 590.897 100 23347 45.22 47.05 3733626 1534927 3733626 1534927 45.61 45.89 3733626 1548395 3733626 1496993 93948698 30.70 1.45 0 3.69 0 0.11 0 0.27 0 0.00 0 4.96 0 3394614 0 100 0 98.84 0 1.51 0 0.02 0 1.16 0 0.01 0 403.43 0 0.19 0 51892 0 3586047 0 132234 0 3882 0 9678 0 0 0 177873 0 123 0 0 0 702 0 98006 0 2574 0 101405 0 90.97 0 3262380 0 8919 99986 11.210449601973 3586047.0 3394614.0 51892.0 132234.0 3882.0 9678.0 0.0 177873.0 3262380.0 94.7 1.4 3.7 0.1 0.3 0.0 5.0 91.0 50 50 50.00 38 179302350 31.0 18.3 18.4 32.3 0.0 37.7 25.3 smartseq 1485395 SRR4250552 SRP090061 SRS1698228 SRX2170553 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316771: 54Dp1_G01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316771 GSM2316771 54Dp1_G01_smart-seq 144366600 1443666 2016-09-30 15:56:31 65178041 144366600 1443666 2 1443666 index:0,count:1443666,average:50,stdev:0|index:1,count:1443666,average:50,stdev:0 GSM2316771_r1 6.0 3.18 0.14 119718474 163110872 109806117 152375837 136.25 138.77 1270967 1019284 261.440 2609.655 81 3979 80.01 87.52 1597028 1016943 1597028 1016943 81.15 83.18 1597028 1031373 1597028 966532 7882474 6.58 2.13 0 7.55 0 0.11 0 0.05 0 0.00 0 11.80 0 1270967 0 100 0 98.47 0 1.23 0 0.01 0 1.17 0 0.01 0 247.49 0 0.21 0 30735 0 1443666 0 109005 0 1639 0 660 0 0 0 170400 0 100 0 0 0 1147 0 144887 0 941 0 147075 0 80.49 0 1161962 0 24460 149142 6.097383483238 1443666.0 1270967.0 30735.0 109005.0 1639.0 660.0 0.0 170400.0 1161962.0 88.0 2.1 7.6 0.1 0.0 0.0 11.8 80.5 50 50 50.00 28 72183300 26.3 22.6 22.7 28.4 0.0 36.5 23.3 smartseq 1485396 SRR4251552 SRP090061 SRS1699228 SRX2171553 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317771: 26Dp3_C04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317771 GSM2317771 26Dp3_C04_smart-seq 437903400 4379034 2016-09-30 15:56:31 297286853 437903400 4379034 2 4379034 index:0,count:4379034,average:50,stdev:0|index:1,count:4379034,average:50,stdev:0 GSM2317771_r1 3.67 3.64 0.11 375084309 434491407 362001109 420958183 115.84 116.29 4181705 3919656 152.883 783.994 81 27147 46.52 48.29 4529454 1945464 4529454 1945464 46.88 46.84 4529454 1960234 4529454 1886764 119877999 31.96 1.71 0 3.50 0 0.13 0 0.27 0 0.00 0 4.11 0 4181705 0 100 0 98.77 0 1.49 0 0.02 0 1.20 0 0.01 0 404.22 0 0.19 0 74972 0 4379034 0 153309 0 5650 0 11643 0 0 0 180036 0 179 0 0 0 1687 0 213337 0 4720 0 219923 0 91.99 0 4028396 0 22912 217327 9.485291550279 4379034.0 4181705.0 74972.0 153309.0 5650.0 11643.0 0.0 180036.0 4028396.0 95.5 1.7 3.5 0.1 0.3 0.0 4.1 92.0 50 50 50.00 38 218951700 30.2 19.4 19.4 30.9 0.0 37.7 27.3 smartseq 1485426 SRR4250553 SRP090061 SRS1698230 SRX2170554 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316772: 54Dp1_G02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316772 GSM2316772 54Dp1_G02_smart-seq 94162900 941629 2016-09-30 15:56:31 43098024 94162900 941629 2 941629 index:0,count:941629,average:50,stdev:0|index:1,count:941629,average:50,stdev:0 GSM2316772_r1 9.22 3.13 0.07 81190437 111820141 75467604 105874557 137.73 140.29 853616 655957 285.152 3173.862 99 2174 82.62 89.13 1053607 705275 1053607 705275 83.24 85.42 1053607 710547 1053607 675942 4707946 5.80 1.89 0 6.62 0 0.09 0 0.05 0 0.00 0 9.21 0 853616 0 100 0 98.58 0 1.22 0 0.01 0 1.16 0 0.01 0 225.99 0 0.22 0 17839 0 941629 0 62309 0 837 0 426 0 0 0 86750 0 81 0 0 0 877 0 106422 0 517 0 107897 0 84.04 0 791307 0 33622 109509 3.257063827256 941629.0 853616.0 17839.0 62309.0 837.0 426.0 0.0 86750.0 791307.0 90.7 1.9 6.6 0.1 0.0 0.0 9.2 84.0 50 50 50.00 28 47081450 26.3 22.9 23.0 27.8 0.0 36.5 23.9 smartseq 1485427 SRR4251553 SRP090061 SRS1699229 SRX2171554 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317772: 26Dp3_C05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317772 GSM2317772 26Dp3_C05_smart-seq 482492400 4824924 2016-09-30 15:56:31 326325629 482492400 4824924 2 4824924 index:0,count:4824924,average:50,stdev:0|index:1,count:4824924,average:50,stdev:0 GSM2317772_r1 5.17 3.13 0.31 418761228 486132709 404048212 470754689 116.09 116.51 4612266 4340406 154.850 729.716 100 30255 46.5 48.27 4974031 2144612 4974031 2144612 46.9 46.8 4974031 2163149 4974031 2079636 133010451 31.76 1.56 0 3.50 0 0.11 0 0.32 0 0.00 0 3.97 0 4612266 0 100 0 98.88 0 1.49 0 0.02 0 1.21 0 0.01 0 598.96 0 0.19 0 75125 0 4824924 0 169014 0 5419 0 15640 0 0 0 191599 0 279 0 0 0 1674 0 212679 0 4305 0 218937 0 92.09 0 4443252 0 17287 218950 12.665586857176 4824924.0 4612266.0 75125.0 169014.0 5419.0 15640.0 0.0 191599.0 4443252.0 95.6 1.6 3.5 0.1 0.3 0.0 4.0 92.1 50 50 50.00 38 241246200 30.6 18.9 19.1 31.4 0.0 37.7 27.0 smartseq 1485458 SRR4250554 SRP090061 SRS1698231 SRX2170555 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316773: 54Dp1_G03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316773 GSM2316773 54Dp1_G03_smart-seq 88129000 881290 2016-09-30 15:56:31 40051778 88129000 881290 2 881290 index:0,count:881290,average:50,stdev:0|index:1,count:881290,average:50,stdev:0 GSM2316773_r1 6.49 3.07 0.13 73601452 97975839 67782191 92186376 133.12 136.0 777081 624701 268.074 2751.176 111 2256 77.96 84.91 990064 605785 990064 605785 78.94 81.26 990064 613406 990064 579705 5913260 8.03 1.83 0 7.22 0 0.11 0 0.08 0 0.00 0 11.63 0 777081 0 100 0 98.56 0 1.27 0 0.01 0 1.15 0 0.01 0 186.63 0 0.22 0 16089 0 881290 0 63668 0 975 0 696 0 0 0 102538 0 104 0 0 0 627 0 85179 0 504 0 86414 0 80.95 0 713413 0 23984 88548 3.691961307538 881290.0 777081.0 16089.0 63668.0 975.0 696.0 0.0 102538.0 713413.0 88.2 1.8 7.2 0.1 0.1 0.0 11.6 81.0 50 50 50.00 28 44064500 26.5 22.5 22.5 28.5 0.0 36.4 23.2 smartseq 1485459 SRR4251554 SRP090061 SRS1699230 SRX2171555 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317773: 26Dp3_C06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317773 GSM2317773 26Dp3_C06_smart-seq 416359800 4163598 2016-09-30 15:56:31 281762780 416359800 4163598 2 4163598 index:0,count:4163598,average:50,stdev:0|index:1,count:4163598,average:50,stdev:0 GSM2317773_r1 2.34 2.98 0.16 354479716 407075586 340674357 392848193 114.84 115.31 3957996 3764432 149.972 680.787 100 27536 45.95 47.9 4343220 1818860 4343220 1818860 46.7 46.69 4343220 1848330 4343220 1772923 115305254 32.53 1.59 0 3.86 0 0.13 0 0.30 0 0.00 0 4.51 0 3957996 0 100 0 98.85 0 1.51 0 0.03 0 1.16 0 0.01 0 405.11 0 0.19 0 66278 0 4163598 0 160638 0 5445 0 12516 0 0 0 187641 0 96 0 0 0 1216 0 152517 0 4454 0 158283 0 91.20 0 3797358 0 16430 156323 9.514485696896 4163598.0 3957996.0 66278.0 160638.0 5445.0 12516.0 0.0 187641.0 3797358.0 95.1 1.6 3.9 0.1 0.3 0.0 4.5 91.2 50 50 50.00 38 208179900 31.0 18.5 18.6 31.9 0.0 37.7 26.2 smartseq 1485491 SRR4250555 SRP090061 SRS1698232 SRX2170556 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316774: 54Dp1_G04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316774 GSM2316774 54Dp1_G04_smart-seq 171237700 1712377 2016-09-30 15:56:31 81205236 171237700 1712377 2 1712377 index:0,count:1712377,average:50,stdev:0|index:1,count:1712377,average:50,stdev:0 GSM2316774_r1 10.37 2.82 0.12 141156750 191056132 130663927 180509769 135.35 138.15 1520277 1275698 227.149 2151.145 81 5899 77.48 84.03 1905981 1177849 1905981 1177849 78.24 80.44 1905981 1189412 1905981 1127569 11501155 8.15 1.89 0 6.93 0 0.12 0 0.09 0 0.00 0 11.02 0 1520277 0 100 0 98.54 0 1.19 0 0.01 0 1.14 0 0.01 0 205.49 0 0.24 0 32287 0 1712377 0 118585 0 1980 0 1460 0 0 0 188660 0 132 0 0 0 1315 0 158625 0 863 0 160935 0 81.86 0 1401692 0 33854 163391 4.826342529686 1712377.0 1520277.0 32287.0 118585.0 1980.0 1460.0 0.0 188660.0 1401692.0 88.8 1.9 6.9 0.1 0.1 0.0 11.0 81.9 50 50 50.00 28 85618850 26.8 22.5 22.3 28.5 0.0 36.4 23.6 smartseq 1485492 SRR4251555 SRP090061 SRS1699232 SRX2171556 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317774: 26Dp3_C07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317774 GSM2317774 26Dp3_C07_smart-seq 564194200 5641942 2016-09-30 15:56:31 381276972 564194200 5641942 2 5641942 index:0,count:5641942,average:50,stdev:0|index:1,count:5641942,average:50,stdev:0 GSM2317774_r1 2.1 3.7 0.3 479368999 541897097 463091342 525221963 113.04 113.42 5377713 5132884 146.623 653.146 81 37339 41.31 42.83 5801633 2221322 5801633 2221322 41.69 41.6 5801633 2241777 5801633 2157672 164941137 34.41 1.71 0 3.39 0 0.11 0 0.24 0 0.00 0 4.34 0 5377713 0 100 0 98.82 0 1.51 0 0.02 0 1.20 0 0.01 0 332.97 0 0.18 0 96203 0 5641942 0 191233 0 6125 0 13465 0 0 0 244639 0 245 0 0 0 1837 0 193823 0 5616 0 201521 0 91.93 0 5186480 0 19532 198044 10.139463444604 5641942.0 5377713.0 96203.0 191233.0 6125.0 13465.0 0.0 244639.0 5186480.0 95.3 1.7 3.4 0.1 0.2 0.0 4.3 91.9 50 50 50.00 38 282097100 31.0 18.5 18.5 32.0 0.0 37.7 26.5 smartseq 1485524 SRR4250556 SRP090061 SRS1698233 SRX2170557 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316775: 54Dp1_G05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316775 GSM2316775 54Dp1_G05_smart-seq 106238400 1062384 2016-09-30 15:56:31 47517445 106238400 1062384 2 1062384 index:0,count:1062384,average:50,stdev:0|index:1,count:1062384,average:50,stdev:0 GSM2316775_r1 4.75 3.25 0.16 89085554 116599356 83348475 110914198 130.88 133.07 942852 769111 250.934 2471.140 100 3031 76.34 81.81 1138920 719795 1138920 719795 76.72 78.41 1138920 723378 1138920 689832 8979438 10.08 1.65 0 5.93 0 0.10 0 0.05 0 0.00 0 11.10 0 942852 0 100 0 98.77 0 1.28 0 0.01 0 1.15 0 0.01 0 424.95 0 0.20 0 17527 0 1062384 0 63021 0 1049 0 578 0 0 0 117905 0 108 0 0 0 765 0 99834 0 616 0 101323 0 82.82 0 879831 0 28872 103318 3.578484344694 1062384.0 942852.0 17527.0 63021.0 1049.0 578.0 0.0 117905.0 879831.0 88.7 1.6 5.9 0.1 0.1 0.0 11.1 82.8 50 50 50.00 28 53119200 27.4 21.7 21.7 29.2 0.0 36.5 23.4 smartseq 1485525 SRR4251556 SRP090061 SRS1699231 SRX2171557 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317775: 26Dp3_C08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317775 GSM2317775 26Dp3_C08_smart-seq 290082600 2900826 2016-09-30 15:56:31 197817567 290082600 2900826 2 2900826 index:0,count:2900826,average:50,stdev:0|index:1,count:2900826,average:50,stdev:0 GSM2317775_r1 3.5 2.88 0.2 258010615 294932677 249763853 286575781 114.31 114.74 2778500 2623968 175.101 816.278 100 16503 40.0 41.37 2994246 1111411 2994246 1111411 40.3 40.16 2994246 1119719 2994246 1078959 95675657 37.08 1.42 0 3.17 0 0.13 0 0.24 0 0.00 0 3.85 0 2778500 0 100 0 99.05 0 1.51 0 0.03 0 1.18 0 0.01 0 316.45 0 0.20 0 41125 0 2900826 0 91874 0 3898 0 6845 0 0 0 111583 0 189 0 0 0 925 0 101207 0 2976 0 105297 0 92.62 0 2686626 0 19859 104668 5.270557429881 2900826.0 2778500.0 41125.0 91874.0 3898.0 6845.0 0.0 111583.0 2686626.0 95.8 1.4 3.2 0.1 0.2 0.0 3.8 92.6 50 50 50.00 38 145041300 30.9 18.6 18.8 31.7 0.0 37.6 26.1 smartseq 1485555 SRR4250557 SRP090061 SRS1698234 SRX2170558 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316776: 54Dp1_G06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316776 GSM2316776 54Dp1_G06_smart-seq 209467100 2094671 2016-09-30 15:56:31 91863077 209467100 2094671 2 2094671 index:0,count:2094671,average:50,stdev:0|index:1,count:2094671,average:50,stdev:0 GSM2316776_r1 3.72 2.96 0.18 174569579 230629120 160773453 216588551 132.11 134.72 1892110 1593795 216.297 1901.545 69 8065 76.92 83.83 2363256 1455408 2363256 1455408 77.68 79.6 2363256 1469770 2363256 1381965 14928053 8.55 1.98 0 7.45 0 0.10 0 0.07 0 0.00 0 9.50 0 1892110 0 100 0 98.59 0 1.29 0 0.01 0 1.16 0 0.01 0 260.03 0 0.21 0 41479 0 2094671 0 156010 0 2156 0 1426 0 0 0 198979 0 146 0 0 0 1643 0 198857 0 1064 0 201710 0 82.88 0 1736100 0 35703 205212 5.747752289724 2094671.0 1892110.0 41479.0 156010.0 2156.0 1426.0 0.0 198979.0 1736100.0 90.3 2.0 7.4 0.1 0.1 0.0 9.5 82.9 50 50 50.00 28 104733550 27.4 22.0 21.9 28.8 0.0 36.6 24.4 smartseq 1485556 SRR4251557 SRP090061 SRS1699235 SRX2171558 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317776: 26Dp3_C09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317776 GSM2317776 26Dp3_C09_smart-seq 111725400 1117254 2016-09-30 15:56:31 76139394 111725400 1117254 2 1117254 index:0,count:1117254,average:50,stdev:0|index:1,count:1117254,average:50,stdev:0 GSM2317776_r1 2.75 3.74 0.28 99150176 113712328 95418569 110029622 114.69 115.31 1063626 998080 187.390 972.382 108 5599 41.75 43.43 1162014 444056 1162014 444056 42.06 42.16 1162014 447308 1162014 431045 34656524 34.95 1.45 0 3.69 0 0.15 0 0.21 0 0.00 0 4.44 0 1063626 0 100 0 99.00 0 1.50 0 0.03 0 1.17 0 0.01 0 287.29 0 0.21 0 16250 0 1117254 0 41211 0 1731 0 2296 0 0 0 49601 0 32 0 0 0 334 0 40021 0 1318 0 41705 0 91.51 0 1022415 0 13041 41158 3.156046315467 1117254.0 1063626.0 16250.0 41211.0 1731.0 2296.0 0.0 49601.0 1022415.0 95.2 1.5 3.7 0.2 0.2 0.0 4.4 91.5 50 50 50.00 38 55862700 30.4 19.0 19.2 31.4 0.0 37.6 25.5 smartseq 1485586 SRR4250558 SRP090061 SRS1698237 SRX2170559 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316777: 54Dp1_G07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316777 GSM2316777 54Dp1_G07_smart-seq 277561800 2775618 2016-09-30 15:56:31 118750327 277561800 2775618 2 2775618 index:0,count:2775618,average:50,stdev:0|index:1,count:2775618,average:50,stdev:0 GSM2316777_r1 7.2 2.94 0.17 229682659 306133596 215510506 291209401 133.29 135.13 2538170 2222581 184.863 1560.276 69 14259 77.44 82.85 2997836 1965671 2997836 1965671 78.1 79.71 2997836 1982306 2997836 1891176 21968899 9.56 1.85 0 5.97 0 0.08 0 0.07 0 0.00 0 8.40 0 2538170 0 100 0 98.62 0 1.31 0 0.01 0 1.15 0 0.01 0 312.26 0 0.18 0 51369 0 2775618 0 165618 0 2351 0 1994 0 0 0 233103 0 173 0 0 0 1723 0 230362 0 1181 0 233439 0 85.48 0 2372552 0 39829 235610 5.915538928921 2775618.0 2538170.0 51369.0 165618.0 2351.0 1994.0 0.0 233103.0 2372552.0 91.4 1.9 6.0 0.1 0.1 0.0 8.4 85.5 50 50 50.00 28 138780900 28.9 20.5 20.3 30.4 0.0 36.7 24.9 smartseq 1485587 SRR4251558 SRP090061 SRS1699234 SRX2171559 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317777: 26Dp3_C10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317777 GSM2317777 26Dp3_C10_smart-seq 117811500 1178115 2016-09-30 15:56:31 82111319 117811500 1178115 2 1178115 index:0,count:1178115,average:50,stdev:0|index:1,count:1178115,average:50,stdev:0 GSM2317777_r1 3.3 3.11 0.12 104127967 119267771 100022584 115289758 114.54 115.26 1099693 1015249 220.053 1237.245 110 4595 41.61 43.36 1223759 457535 1223759 457535 42.07 42.06 1223759 462674 1223759 443816 36398729 34.96 1.71 0 3.77 0 0.15 0 0.27 0 0.00 0 6.24 0 1099693 0 100 0 98.92 0 1.52 0 0.03 0 1.21 0 0.01 0 235.62 0 0.25 0 20096 0 1178115 0 44450 0 1825 0 3139 0 0 0 73458 0 43 0 0 0 475 0 45733 0 1537 0 47788 0 89.57 0 1055243 0 14731 47508 3.225035639128 1178115.0 1099693.0 20096.0 44450.0 1825.0 3139.0 0.0 73458.0 1055243.0 93.3 1.7 3.8 0.2 0.3 0.0 6.2 89.6 50 50 50.00 38 58905750 29.4 19.8 19.9 30.9 0.0 37.3 23.9 smartseq 1485617 SRR4250559 SRP090061 SRS1698235 SRX2170560 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316778: 54Dp1_G08_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316778 GSM2316778 54Dp1_G08_smart-seq 217975300 2179753 2016-09-30 15:56:31 93950423 217975300 2179753 2 2179753 index:0,count:2179753,average:50,stdev:0|index:1,count:2179753,average:50,stdev:0 GSM2316778_r1 12.02 3.03 0.12 183327674 251408511 169347843 236005998 137.14 139.36 2018287 1764486 191.021 1492.163 69 11705 75.27 81.91 2461646 1519084 2461646 1519084 77.46 78.94 2461646 1563339 2461646 1463973 18307125 9.99 1.96 0 7.52 0 0.13 0 0.08 0 0.00 0 7.19 0 2018287 0 100 0 98.63 0 1.26 0 0.01 0 1.14 0 0.01 0 313.88 0 0.20 0 42713 0 2179753 0 163813 0 2904 0 1791 0 0 0 156771 0 139 0 0 0 1086 0 182498 0 945 0 184668 0 85.08 0 1854474 0 43563 184996 4.246631315566 2179753.0 2018287.0 42713.0 163813.0 2904.0 1791.0 0.0 156771.0 1854474.0 92.6 2.0 7.5 0.1 0.1 0.0 7.2 85.1 50 50 50.00 28 108987650 28.9 20.5 20.5 30.1 0.0 36.7 25.5 smartseq 1485618 SRR4251559 SRP090061 SRS1699237 SRX2171560 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317778: 26Dp3_C11_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317778 GSM2317778 26Dp3_C11_smart-seq 169691400 1696914 2016-09-30 15:56:31 118804843 169691400 1696914 2 1696914 index:0,count:1696914,average:50,stdev:0|index:1,count:1696914,average:50,stdev:0 GSM2317778_r1 3.07 2.81 0.11 150908752 174558006 144651745 168269114 115.67 116.33 1582256 1438505 232.170 1426.103 135 6121 45.85 47.88 1766198 725396 1766198 725396 46.43 46.4 1766198 734705 1766198 703041 48211016 31.95 1.59 0 3.95 0 0.12 0 0.18 0 0.00 0 6.46 0 1582256 0 100 0 98.95 0 1.52 0 0.02 0 1.21 0 0.01 0 254.54 0 0.25 0 26963 0 1696914 0 67099 0 2035 0 2989 0 0 0 109634 0 56 0 0 0 684 0 77007 0 2331 0 80078 0 89.29 0 1515157 0 18855 80098 4.248103951207 1696914.0 1582256.0 26963.0 67099.0 2035.0 2989.0 0.0 109634.0 1515157.0 93.2 1.6 4.0 0.1 0.2 0.0 6.5 89.3 50 50 50.00 38 84845700 28.7 20.5 20.7 30.0 0.0 37.2 24.1 smartseq 1485841 SRR4250560 SRP090061 SRS1698236 SRX2170561 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316779: 54Dp1_G09_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316779 GSM2316779 54Dp1_G09_smart-seq 148834600 1488346 2016-09-30 15:56:31 64509347 148834600 1488346 2 1488346 index:0,count:1488346,average:50,stdev:0|index:1,count:1488346,average:50,stdev:0 GSM2316779_r1 7.82 3.04 0.13 125035356 165204057 117798233 157636812 132.13 133.82 1353635 1147473 203.896 2003.219 78 5991 77.02 81.99 1593272 1042612 1593272 1042612 77.62 78.97 1593272 1050641 1593272 1004182 12319495 9.85 1.67 0 5.51 0 0.09 0 0.08 0 0.00 0 8.88 0 1353635 0 100 0 98.69 0 1.27 0 0.01 0 1.15 0 0.01 0 267.90 0 0.19 0 24818 0 1488346 0 82006 0 1308 0 1225 0 0 0 132178 0 112 0 0 0 1059 0 136964 0 641 0 138776 0 85.44 0 1271629 0 31951 140517 4.397890519859 1488346.0 1353635.0 24818.0 82006.0 1308.0 1225.0 0.0 132178.0 1271629.0 90.9 1.7 5.5 0.1 0.1 0.0 8.9 85.4 50 50 50.00 28 74417300 28.0 21.4 21.3 29.3 0.0 36.6 24.9 smartseq 1485842 SRR4251560 SRP090061 SRS1699236 SRX2171561 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317779: 26Dp3_D01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317779 GSM2317779 26Dp3_D01_smart-seq 68780700 687807 2016-09-30 15:56:31 48894749 68780700 687807 2 687807 index:0,count:687807,average:50,stdev:0|index:1,count:687807,average:50,stdev:0 GSM2317779_r1 3.78 2.44 0.24 59327050 67345744 56347800 64659684 113.52 114.75 627736 585486 222.874 1049.529 116 2711 47.71 50.31 730651 299503 730651 299503 48.46 48.82 730651 304207 730651 290676 17281131 29.13 1.46 0 4.71 0 0.10 0 0.20 0 0.00 0 8.43 0 627736 0 100 0 98.90 0 1.47 0 0.02 0 1.18 0 0.01 0 154.76 0 0.27 0 10016 0 687807 0 32384 0 696 0 1365 0 0 0 58010 0 21 0 0 0 243 0 22651 0 579 0 23494 0 86.56 0 595352 0 8947 23636 2.641779367386 687807.0 627736.0 10016.0 32384.0 696.0 1365.0 0.0 58010.0 595352.0 91.3 1.5 4.7 0.1 0.2 0.0 8.4 86.6 50 50 50.00 38 34390350 29.4 19.6 19.7 31.3 0.0 37.1 23.0 smartseq 1485872 SRR4250561 SRP090061 SRS1698238 SRX2170562 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316780: 54Dp1_G10_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316780 GSM2316780 54Dp1_G10_smart-seq 129337700 1293377 2016-09-30 15:56:31 58043619 129337700 1293377 2 1293377 index:0,count:1293377,average:50,stdev:0|index:1,count:1293377,average:50,stdev:0 GSM2316780_r1 5.23 3.05 0.12 110270392 147412462 101595156 138406151 133.68 136.23 1164970 929467 255.313 2711.683 89 3548 78.95 85.93 1462791 919788 1462791 919788 79.93 82.05 1462791 931155 1462791 878265 8667678 7.86 1.76 0 7.31 0 0.08 0 0.07 0 0.00 0 9.78 0 1164970 0 100 0 98.70 0 1.24 0 0.01 0 1.14 0 0.01 0 232.81 0 0.21 0 22761 0 1293377 0 94573 0 971 0 970 0 0 0 126466 0 83 0 0 0 1082 0 138179 0 722 0 140066 0 82.76 0 1070397 0 31889 143152 4.489071466650 1293377.0 1164970.0 22761.0 94573.0 971.0 970.0 0.0 126466.0 1070397.0 90.1 1.8 7.3 0.1 0.1 0.0 9.8 82.8 50 50 50.00 28 64668850 26.6 22.6 22.7 28.1 0.0 36.5 23.8 smartseq 1485873 SRR4251561 SRP090061 SRS1699242 SRX2171562 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317780: 26Dp3_D02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317780 GSM2317780 26Dp3_D02_smart-seq 264045800 2640458 2016-09-30 15:56:31 183797863 264045800 2640458 2 2640458 index:0,count:2640458,average:50,stdev:0|index:1,count:2640458,average:50,stdev:0 GSM2317780_r1 1.91 3.52 0.2 229893557 261121147 221290524 252340430 113.58 114.03 2510665 2360060 168.080 853.210 100 15393 44.3 46.08 2747759 1112275 2747759 1112275 44.68 44.63 2747759 1121823 2747759 1077416 76423940 33.24 1.52 0 3.66 0 0.14 0 0.20 0 0.00 0 4.58 0 2510665 0 100 0 98.91 0 1.48 0 0.02 0 1.20 0 0.01 0 475.28 0 0.22 0 40027 0 2640458 0 96703 0 3578 0 5238 0 0 0 120977 0 99 0 0 0 818 0 102805 0 2331 0 106053 0 91.42 0 2413962 0 18055 105710 5.854887842703 2640458.0 2510665.0 40027.0 96703.0 3578.0 5238.0 0.0 120977.0 2413962.0 95.1 1.5 3.7 0.1 0.2 0.0 4.6 91.4 50 50 50.00 38 132022900 30.6 18.9 19.0 31.4 0.0 37.5 26.0 smartseq 1485904 SRR4251562 SRP090061 SRS1699239 SRX2171563 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317781: 26Dp3_D03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317781 GSM2317781 26Dp3_D03_smart-seq 316253400 3162534 2016-09-30 15:56:31 219394722 316253400 3162534 2 3162534 index:0,count:3162534,average:50,stdev:0|index:1,count:3162534,average:50,stdev:0 GSM2317781_r1 1.4 2.96 0.08 272667530 314796945 263449528 305280401 115.45 115.88 3002316 2826827 157.182 813.380 100 19703 49.37 51.16 3255381 1482112 3255381 1482112 49.55 49.6 3255381 1487687 3255381 1436749 83183655 30.51 1.48 0 3.33 0 0.14 0 0.24 0 0.00 0 4.69 0 3002316 0 100 0 98.89 0 1.52 0 0.02 0 1.18 0 0.01 0 367.26 0 0.20 0 46847 0 3162534 0 105449 0 4288 0 7518 0 0 0 148412 0 93 0 0 0 1069 0 130733 0 2362 0 134257 0 91.60 0 2896867 0 15897 132677 8.346040133358 3162534.0 3002316.0 46847.0 105449.0 4288.0 7518.0 0.0 148412.0 2896867.0 94.9 1.5 3.3 0.1 0.2 0.0 4.7 91.6 50 50 50.00 38 158126700 30.8 18.7 18.8 31.6 0.0 37.6 26.4 smartseq 1485937 SRR4250563 SRP090061 SRS1698240 SRX2170564 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316782: 54Dp1_H01_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316782 GSM2316782 54Dp1_H01_smart-seq 108093800 1080938 2016-09-30 15:56:31 50200119 108093800 1080938 2 1080938 index:0,count:1080938,average:50,stdev:0|index:1,count:1080938,average:50,stdev:0 GSM2316782_r1 6.92 3.28 0.15 91183182 122139085 84713937 115698092 133.95 136.58 968689 769686 267.259 2924.626 80 3024 79.39 85.73 1207579 769017 1207579 769017 79.85 82.06 1207579 773514 1207579 736111 7024317 7.70 1.90 0 6.63 0 0.09 0 0.04 0 0.00 0 10.25 0 968689 0 100 0 98.49 0 1.19 0 0.01 0 1.15 0 0.01 0 228.90 0 0.22 0 20501 0 1080938 0 71633 0 946 0 457 0 0 0 110846 0 66 0 0 0 832 0 111861 0 613 0 113372 0 82.99 0 897056 0 32065 115283 3.595290815531 1080938.0 968689.0 20501.0 71633.0 946.0 457.0 0.0 110846.0 897056.0 89.6 1.9 6.6 0.1 0.0 0.0 10.3 83.0 50 50 50.00 28 54046900 26.3 22.7 22.7 28.3 0.0 36.2 23.0 smartseq 1485938 SRR4251563 SRP090061 SRS1699238 SRX2171564 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317782: 26Dp3_D04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317782 GSM2317782 26Dp3_D04_smart-seq 423502000 4235020 2016-09-30 15:56:31 293298827 423502000 4235020 2 4235020 index:0,count:4235020,average:50,stdev:0|index:1,count:4235020,average:50,stdev:0 GSM2317782_r1 6.24 3.06 0.29 347935623 403096601 334332882 390178399 115.85 116.7 3983310 3796077 137.551 779.337 71 30664 45.93 47.9 4377265 1829524 4377265 1829524 46.52 46.69 4377265 1853047 4377265 1783575 107329894 30.85 1.96 0 3.86 0 0.25 0 0.40 0 0.00 0 5.29 0 3983310 0 100 0 98.56 0 1.45 0 0.02 0 1.21 0 0.01 0 412.06 0 0.22 0 82918 0 4235020 0 163602 0 10777 0 17106 0 0 0 223827 0 167 0 0 0 1241 0 158055 0 3582 0 163045 0 90.19 0 3819708 0 15869 162643 10.249102022812 4235020.0 3983310.0 82918.0 163602.0 10777.0 17106.0 0.0 223827.0 3819708.0 94.1 2.0 3.9 0.3 0.4 0.0 5.3 90.2 50 50 50.00 38 211751000 30.3 19.4 19.3 31.0 0.0 37.6 27.1 smartseq 1485971 SRR4250564 SRP090061 SRS1698242 SRX2170565 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316783: 54Dp1_H02_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316783 GSM2316783 54Dp1_H02_smart-seq 65375800 653758 2016-09-30 15:56:31 29663227 65375800 653758 2 653758 index:0,count:653758,average:50,stdev:0|index:1,count:653758,average:50,stdev:0 GSM2316783_r1 1.27 2.11 0.08 37292970 44294562 30396429 40351609 118.77 132.75 404459 329148 250.213 2640.747 54 2228 66.04 82.87 742517 267113 742517 267113 66.51 78.88 742517 269011 742517 254264 3030417 8.13 1.55 0 12.56 0 0.08 0 0.06 0 0.00 0 38.00 0 404459 0 100 0 98.47 0 1.30 0 0.01 0 1.17 0 0.01 0 98.06 0 0.25 0 10157 0 653758 0 82136 0 525 0 371 0 0 0 248403 0 33 0 0 0 327 0 41692 0 267 0 42319 0 49.30 0 322323 0 18048 41702 2.310616134752 653758.0 404459.0 10157.0 82136.0 525.0 371.0 0.0 248403.0 322323.0 61.9 1.6 12.6 0.1 0.1 0.0 38.0 49.3 50 50 50.00 28 32687900 25.2 23.7 24.0 27.1 0.0 36.3 23.0 smartseq 1485972 SRR4251564 SRP090061 SRS1699243 SRX2171565 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317783: 26Dp3_D05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317783 GSM2317783 26Dp3_D05_smart-seq 298939800 2989398 2016-09-30 15:56:31 207239518 298939800 2989398 2 2989398 index:0,count:2989398,average:50,stdev:0|index:1,count:2989398,average:50,stdev:0 GSM2317783_r1 2.75 3.11 0.33 256886372 288462246 247444007 279315207 112.29 112.88 2834099 2675767 159.582 783.290 100 18168 44.32 46.08 3098826 1256026 3098826 1256026 44.82 44.82 3098826 1270272 3098826 1221779 81409239 31.69 1.59 0 3.62 0 0.26 0 0.35 0 0.00 0 4.59 0 2834099 0 100 0 98.86 0 1.48 0 0.02 0 1.17 0 0.01 0 384.35 0 0.21 0 47404 0 2989398 0 108157 0 7814 0 10316 0 0 0 137169 0 46 0 0 0 917 0 114835 0 2830 0 118628 0 91.19 0 2725942 0 16588 117913 7.108331323849 2989398.0 2834099.0 47404.0 108157.0 7814.0 10316.0 0.0 137169.0 2725942.0 94.8 1.6 3.6 0.3 0.3 0.0 4.6 91.2 50 50 50.00 38 149469900 30.7 18.8 19.0 31.5 0.0 37.6 26.5 smartseq 1486004 SRR4250565 SRP090061 SRS1698241 SRX2170566 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316784: 54Dp1_H03_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316784 GSM2316784 54Dp1_H03_smart-seq 89336800 893368 2016-09-30 15:56:31 41209887 89336800 893368 2 893368 index:0,count:893368,average:50,stdev:0|index:1,count:893368,average:50,stdev:0 GSM2316784_r1 3.73 3.1 0.11 71807674 92723546 67450344 88456982 129.13 131.14 761594 613146 259.081 2890.924 99 2403 75.1 80.16 920509 571921 920509 571921 74.83 76.52 920509 569893 920509 545988 8159015 11.36 1.69 0 5.39 0 0.11 0 0.07 0 0.00 0 14.57 0 761594 0 100 0 98.59 0 1.25 0 0.01 0 1.18 0 0.01 0 178.67 0 0.21 0 15132 0 893368 0 48108 0 1016 0 599 0 0 0 130159 0 76 0 0 0 658 0 85356 0 523 0 86613 0 79.86 0 713486 0 25811 87722 3.398628491728 893368.0 761594.0 15132.0 48108.0 1016.0 599.0 0.0 130159.0 713486.0 85.2 1.7 5.4 0.1 0.1 0.0 14.6 79.9 50 50 50.00 28 44668400 26.2 22.7 22.6 28.5 0.0 36.2 22.7 smartseq 1486005 SRR4251565 SRP090061 SRS1699240 SRX2171566 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317784: 26Dp3_D06_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317784 GSM2317784 26Dp3_D06_smart-seq 264475500 2644755 2016-09-30 15:56:31 183837798 264475500 2644755 2 2644755 index:0,count:2644755,average:50,stdev:0|index:1,count:2644755,average:50,stdev:0 GSM2317784_r1 2.13 2.63 0.2 231833884 258640325 224816159 252015807 111.56 112.1 2525194 2402149 164.771 809.497 103 16224 39.99 41.28 2708836 1009778 2708836 1009778 40.07 40.13 2708836 1011943 2708836 981649 83445157 35.99 1.43 0 2.99 0 0.12 0 0.25 0 0.00 0 4.14 0 2525194 0 100 0 99.00 0 1.52 0 0.02 0 1.17 0 0.01 0 453.39 0 0.21 0 37861 0 2644755 0 79006 0 3298 0 6699 0 0 0 109564 0 39 0 0 0 704 0 84477 0 2840 0 88060 0 92.49 0 2446188 0 15659 86638 5.532792643208 2644755.0 2525194.0 37861.0 79006.0 3298.0 6699.0 0.0 109564.0 2446188.0 95.5 1.4 3.0 0.1 0.3 0.0 4.1 92.5 50 50 50.00 38 132237750 30.9 18.6 18.8 31.7 0.0 37.5 26.2 smartseq 1486035 SRR4250566 SRP090061 SRS1698243 SRX2170567 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316785: 54Dp1_H04_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316785 GSM2316785 54Dp1_H04_smart-seq 112373800 1123738 2016-09-30 15:56:31 51006510 112373800 1123738 2 1123738 index:0,count:1123738,average:50,stdev:0|index:1,count:1123738,average:50,stdev:0 GSM2316785_r1 3.41 3.37 0.13 92487647 117539901 86271494 111814048 127.09 129.61 988821 802703 247.614 2813.483 54 3422 73.22 78.72 1221142 723994 1221142 723994 73.51 75.38 1221142 726903 1221142 693224 10964949 11.86 1.84 0 6.15 0 0.11 0 0.08 0 0.00 0 11.82 0 988821 0 100 0 98.52 0 1.27 0 0.01 0 1.16 0 0.01 0 269.70 0 0.21 0 20674 0 1123738 0 69136 0 1250 0 888 0 0 0 132779 0 75 0 0 0 869 0 110693 0 598 0 112235 0 81.84 0 919685 0 27987 114343 4.085575445743 1123738.0 988821.0 20674.0 69136.0 1250.0 888.0 0.0 132779.0 919685.0 88.0 1.8 6.2 0.1 0.1 0.0 11.8 81.8 50 50 50.00 28 56186900 26.4 22.7 22.5 28.5 0.0 36.3 23.2 smartseq 1486036 SRR4251566 SRP090061 SRS1699241 SRX2171567 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2317785: 26Dp3_D07_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;26|source_name;;cultured embryonic stem cells GEO Accession;;GSM2317785 GSM2317785 26Dp3_D07_smart-seq 339993900 3399939 2016-09-30 15:56:31 236238327 339993900 3399939 2 3399939 index:0,count:3399939,average:50,stdev:0|index:1,count:3399939,average:50,stdev:0 GSM2317785_r1 4.19 3.29 0.17 292516099 343435452 281342371 331817979 117.41 117.94 3245032 3078686 154.225 734.027 100 21705 46.39 48.32 3544228 1505524 3544228 1505524 46.89 46.89 3544228 1521571 3544228 1461105 92491475 31.62 1.61 0 3.80 0 0.13 0 0.22 0 0.00 0 4.21 0 3245032 0 100 0 98.86 0 1.50 0 0.02 0 1.20 0 0.01 0 437.14 0 0.21 0 54713 0 3399939 0 129277 0 4410 0 7423 0 0 0 143074 0 167 0 0 0 946 0 122884 0 3032 0 127029 0 91.64 0 3115755 0 18566 128693 6.931649251320 3399939.0 3245032.0 54713.0 129277.0 4410.0 7423.0 0.0 143074.0 3115755.0 95.4 1.6 3.8 0.1 0.2 0.0 4.2 91.6 50 50 50.00 38 169996950 30.7 18.9 19.0 31.4 0.0 37.5 26.7 smartseq 1486067 SRR4250567 SRP090061 SRS1698244 SRX2170568 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. Overall design: The transcriptomes of 1846 single cells were profiled by SmartSeq2 at different timepoints throughout a 54-day differentiation protocol that converted H1 human embryonic stem cells to a variety of brain cell types. Some cells were positively labeled by a expression of a barcoded viral transgene to help establish clonality (marked by an "SK"). GSM2316786: 54Dp1_H05_smart-seq; Homo sapiens; RNA-Seq RNA-Seq TRANSCRIPTOMIC cDNA paired To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy. SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads Illumina HiSeq 2500 control;;FALSE|cre line;;DCX+|days in culture;;54|source_name;;cultured embryonic stem cells GEO Accession;;GSM2316786 GSM2316786 54Dp1_H05_smart-seq 102162400 1021624 2016-09-30 15:56:31 46547795 102162400 1021624 2 1021624 index:0,count:1021624,average:50,stdev:0|index:1,count:1021624,average:50,stdev:0 GSM2316786_r1 5.56 3.27 0.19 85560262 112656066 79888824 107099219 131.67 134.06 905289 719242 262.221 3107.982 81 2848 80.31 86.25 1108744 726997 1108744 726997 80.27 82.38 1108744 726716 1108744 694387 6203193 7.25 1.75 0 6.10 0 0.08 0 0.07 0 0.00 0 11.24 0 905289 0 100 0 98.62 0 1.26 0 0.01 0 1.15 0 0.01 0 204.32 0 0.21 0 17855 0 1021624 0 62349 0 827 0 698 0 0 0 114810 0 62 0 0 0 725 0 106735 0 686 0 108208 0 82.51 0 842940 0 28632 109519 3.825055881531 1021624.0 905289.0 17855.0 62349.0 827.0 698.0 0.0 114810.0 842940.0 88.6 1.7 6.1 0.1 0.1 0.0 11.2 82.5 50 50 50.00 28 51081200 26.3 22.8 22.6 28.3 0.0 36.2 23.1 smartseq 1486068 SRR4251567 SRP090061 SRS1699244 SRX2171568 SRA472738 GEO REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq] During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development. During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered SOX2Cit/+ and DCXCit/Y hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types cons