GSM9378647: UN-SCC680 b2M KO tumor, anti-PD1 + DR-18; Mus musculus; RNA-Seq
tumor tissue was mechanically dissociated and incubated at 37˚C in digestion buffer consisting of RPMI + 2% heat-inactivated fetal bovine serum + 0.5 mg/mL collagenase (Sigma, C5138) + 1 μg/mL DNase I (Sigma, D5025) for 45 minutes. Tissue was subsequently passed through a 70 μM nylon mesh to create single-cell suspensions. Tissue was stained with Live/Dead Fixable Red for dead cell exclusion and the following antibodies: CD45 (30-F11), CD4 (GK1.5), CD8a (53-6.7), CD49b (DX5). Cells were sorted with a BD FACSAria for CD45+CD4/CD8/CD49b+ T and NK cells, CD45+CD4/CD8/CD49b- other immune cells, and CD45- tumor and stromal cells. The three cell populations (T/NK cells, other immune, tumor and stroma) for each individual tumor were mixed at a 2:1:1 ratio. Cells from five tumors per treatment condition were pooled in equal number, and 10,000 cells per group were loaded onto the 10X Genomics Chromium System. Cells for the αPD-1 + DR-18 group were selected from five tumors that were responding at the time of tissue collection, as determined by a reduction in tumor volume from the previous measurement. Library preparation was performed using 10X Genomics reagents according to manufacturer's specifications by the Yale Center for Genome Analysis (YCGA). After passing quality control, libraries were sequenced on the NovaSeq 6000 platform by the YCGA, targeting 50,000 reads per sample.