GSM5851295: DN2; Homo sapiens; RNA-Seq
For the isolation of thymocyte subsets, thymocytes from six donors were pooled. To isolate DN1, DN2, and DN3 thymocytes, CD34 magnetic beads (CD34 MicroBead Kit UltraPure, Miltenyi Biotec) were used to enrich for CD34+ thymocytes after thawing, pooling, and Ficoll density separation. Cell suspensions were barcoded through the 10x Chromium Single Cell platform using Chromium Single Cell 5' Library, from Lin-CD34+CD38-CD1a- (DN1), Lin-CD34+CD38+CD1a- (DN2), Lin-CD34+CD38+CD1a+ (DN3) (Sort 1), and CD34-CD3lowCD4+ (ISP), CD34-CD3-CD4+CD8+ (DP CD3-), CD34-CD3+CD4+CD8+ (DP CD3+),, CD3-CD4+CD8- (CD4SP), CD3+CD4-CD8+ (CD8SP) (Sort 2) and used to generate 8 single-cell 5′ gene expression libraries. The loaded cell numbers range from 300-500,000 aiming for 5000 single cells per reaction. All libraries were sequenced on an Illumina HiSeq 4000 (paired-end 75bp reads) to an average depth of 50.000 reads per cell, resulting in 8 datasets (for each developmental thymocyte subset). Single-cell TCRαβ sequencing libraries were generated using the Chromium Single Cell 5′ Library Construction kit (PN-1000020, 10X Genomics) and Chromium Single Cell V(D)J Enrichment Kit for human T cell (PN-100005, 10X Genomics).  Single-cell TCRγδ sequencing libraries were generated using the Chromium Single Cell 5′ Library construction kit (PN-1000020, 10X Genomics) and custom primer sets. Pooled libraries were sequenced on an Illumina HiSeq4000 (paired-end 150 bp reads) aiming at an average depth of 5000 read pairs per cell.