GSM8223089: Male, 5 months (T096_mRNA); Homo sapiens; RNA-Seq
Briefly, fresh human thymus was minced into small pieces and digested in warm dissociation media (2mg/ml Stemxyme, 6.3 U/mL DNaseI, 1.5% BSA in RPMI) for two 30 min intervals. An additional 8 ml of dissociation cocktail was added for a final 30 min digestion. Cells from the third digestion were collected and depleted of CD45+ cells using the Miltenyi LD column prep kit. Cells were then sorted based on live cells and CD45 expression. For some samples fresh thymocytes were enriched for CD25+ cells and and CD8- cells using StemCell Technologies EasySep kits. Cells were recombined in a 3:2 ratio (CD45-:CD45+) or 2:2:1 ratio (CD45-:CD45+:CD25+) prior to library preparation. Library was prepared using Chromium Single Cell 5ʹ v2 next gem kit and protocol (10x Genomics). All samples were prepared in accordance to the 10X user guidelines (cite user guide).