GSM8488933: TEPs, iPSC-derived, day 63; Homo sapiens; RNA-Seq
To isolate human thymic stromal cells, thymi were digested using 100 µg/ml Liberase TM and 200 µg/ml DNase I in RPMI 1640 with 1% FBS. Cells were resuspended in 83% v/v Percoll in PBS, and a lighter layer of 47% v/v Percoll in PBS and another layer of only PBS were overlaid. After centrifugation, the enriched stromal cell layer was collected and resuspended in buffer containing 1% FBS and 5 mM EDTA in PBS. Cells were incubated for 15 min on ice with 50 µl MojoSort Human CD45 Nanobeads per 1 x 10^8 cells in 1 ml buffer. After one wash, CD45+ cells were depleted on a MagniSort Magnet and the stromal fraction was frozen for future use. On the day of cell preparation for scRNA-seq, the frozen thymic stromal cells were defrosted and stained with antibodies at 1:50 dilution for 15 min on ice. After one wash, cells were passed through a cell strainer and analyzed using the BD FACSAria II. The EPCAM-CD45-CD235ab- and EPCAM+CD31-CD45-CD235ab- fractions were sorted and resuspended in 0.04% BSA in PBS, after which they were mixed 1:2 to enrich for TECs while also retaining other heterogeneous stromal cells. For the iPSC-derived cells, dissociation was performed using Accutase or Accumax, after which cells were washed in buffer containing 0.1% BSA in PBS. Cells were passed through a cell strainer and analyzed using the BD FACSAria II. The mCherry-, mCherry+(low), and mCherry+(high) fractions were sorted and resuspended in 0.04% BSA in PBS, after which they were mixed 1:1:2 to enrich for mCherry+ cells. For samples where no mCherry+ cells were detected (days 7, 11, and 18), the entire live fraction was sorted and resuspended in 0.04% BSA in PBS. For the co-cultured primary thymocytes and induced TECs, the organoids were dissociated using Accumax, washed in 1% FBS in PBS, and analyzed using the BD FACSymphony S6. The mCherry- and mCherry+ fractions were sorted and resuspended in 0.04% BSA in PBS, after which they were mixed at a ratio of 1:2. 10,000 cells per sample were applied to the Chromium Next GEM Chip K, after which reverse transcription, cDNA amplification, and library construction were performed using the Chromium Next GEM Single Cell 5' GEM Kit v2, Chromium Next GEM Single Cell 5' Gel Bead Kit v2, Library Construction Kit, and Dual Index Kit TT Set A according to the manufacturer's instructions (10x Genomics).