GSM8487179: LKR13-H tumor, acquired resistance to anti-PD-1 and anti-CTLA-4; Mus musculus; RNA-Seq
Tumor tissue was minced in RPMI1640 with 2% FBS, 200U/ml collagenase IV, and 1 µg/ml DNase and digested at 37°C for 30 min. Samples were filtered through a 70µm cell strainer, incubated with ammonium-chloride-potassium lysing buffer for 3 min, washed with PBS, and resuspended in RPMI1640 with 2% FBS. The resulting single cell suspension was stained with the following antibodies: CD45:PacificBlue (1:500; BioLegend #103126), CD8:PE (1:100; BioLegend #100512), CD4:PE (1:100; BioLegend #100708), and CD3:APC-Cy7 (1:50). Dead cells were excluded using Live/Dead Red Fixable Viability Dye (1:500). CD45+CD3+CD4/8+ T cells, CD45+CD3-CD4/8- other immune cells, and CD45- cancer (and stromal) cells were sorted on a FACSAria (BD) into RPMI-1640 supplemented with 2% FBS. For each individual tumor, the three sorted cell populations – T cells, other immune cells, and CD45- cancer (and stromal) cells – were combined at a 2:1:1 ratio. Library construction was performed using 10x Genomics reagents according to the manufacturer's instructions (3' library prep kit)