GSM9736508: TNFR2-3F10 treated day 2; Mus musculus; RNA-Seq
Harvested tumors were cut into small (2-3 mm) pieces using scalpels. Tissue pieces were enzymatically digested using 0,5 mg/ml Liberase TM (Merck) and 200 kunitz/ml DNAse (Merck) for 2x5 min at 37 degrees with slight vortex in between. Digestion reaction was quenched with 5 mL of RPMI (containing 10% FCS) and cells were filtered through a 70 mm cell-strainer. Viable lymphocytes were isolated using Lympholyte-M Separation Media (Cedarlane) gradients. Lymphocytes were stained with viability dye and CD45 antibody and live CD45+ cells were sorted using a FACS AriaFusion (Becton Dickinson). Cells were counted (NucleoCounter NC-202, ChemoMetec) and fixed according to “10x sample fixation Protocol” from 10x Genomics and kept at -80 °C until analyzed using scRNA- seq. The viable CD45+ cells were sorted using FACS and samples were fixed using 4% paraformaldehyde (PFA) for 20 hours at 4 °C, followed by long-term storage at –80 °C according to '10x Genomics' flex fixation kit recommendations. In short, prior to library preparation, samples were thawed, washed, and hybridized with barcoded probe sets according to the Chromium Next-GEM Flex protocol (10x Genomics). Approximately 10k cells per sample were targeted for capture, and the pooled samples were loaded onto a Chromium X instrument using a Chip Q. The sequencing Libraries were sequenced on an Illumina NovaSeq 6000