GSM5945438: RNA_YUMM-MCJ-KO TILs; Mus musculus; RNA-Seq
GSM5945438
YUMM-sgSCR and MCJ KO were grown subcutaneosuly and at day 21 post implantation, tumour were cut into approximately 1-2 mm3 pieces in the RPMI-1640 medium (Gibco) with 2% fetal bovine serum (FBS, Gibco), and enzymatically digested with Collagenase IV (sigma) and DNase (Roche) for 20 min on a rotor at 37°C. The dissociated cells were subsequently passed through a 70 μm SmartStrainer and centrifuged at 400g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer and incubated at room temperature for 2 min to lyse red blood cells. After washing twice with 1× PBS (Gibco), the cell pellets were re-suspended in sorting buffer (PBS supplemented with 2% FBS). Upon pooling samples (3 controls and 3 MCJ KO) single cell suspensions were stained and FACS sorted for CD3+ CD8+ T cells, CD3+ CD4+  T cells, CD3- CD45+ cells and CD45- cells using 100uM nozzle. A total of 10,000 cells from each sample were used for the 10x Chromium Single cell 5' and VDJ library construction (10x Genomics), according to the manufacturer's instructions. 10x Chromium Single cell 5' + VDJ Library