GSM6212684: Thymus_gd_3scRNAseq; Homo sapiens; RNA-Seq
GSM6212684
Pediatric thymus from a 4 month old child undergoing cardiac surgery was obtained and used according to and with the approval of the Medical Ethical Commission of Ghent University Hospital (Belgium). Thymus tissue was cut into small pieces and digested with 1.6mg/ml collagenase (Gibco, 17104-019) in IMDM medium for 30min at 37°C to generate a single cell suspension. The reaction was quenched with 10% FBS and the thymocyte suspension was passed through a 70μm filter to remove undigested tissue. Cells were frozen in FBS containing 10% DMSO and stored in liquid nitrogen until needed. Upon thawing thymocytes were enriched for cell types of interest (CD34+ cells, ISPs, DPs, TCRγδ+ thymocytes) using bead-based enrichment/depletion and FACS. To obtain DP thymocytes, cells were labelled with antibodies and FACS sorting was used to obtain equal proportions of CD8α+CD4+CD3+ and CD45+CD8α+CD4+CD3- thymocytes. CD34+ cells were obtained through enrichment with CD34 magnetic-activated cell-sorting (MACS) microbeads (Miltenyi, 130-046-703), labelled with anti-CD1a and subsequently FACS sorted to include equal proportions of CD1a+ and CD1a- cells. To enrich ISPs thymocytes were labelled with anti-CD3 (clone OKT3, produced in house) and anti-glycophorin A (clone 10F7MN, produced in-house) and CD3+ and Glycophorin+ cells were subsequently depleted using sheep anti-mouse IgG magnetic Dynabeads (Invitrogen). This was followed by FACS sorting for CD3-CD8α-CD4+ thymocytes. To obtain TCRγδ+ thymocytes, cells were enriched using anti-γδ TCR hapten antibodies and anti-hapten MACS microbeads (Miltenyi, 130-050-701) according to the manufacturer's instructions and subsequently FACS sorted for TCRγδ+CD3+. library construction according to protocol for 10X Genomics Chromium Next GEM Single Cell 3' Kit v3.1