GSM8192615: BM3_C7; Mus musculus; RNA-Seq
Tumor samples were chopped into small pieces with scissors in digest media containing collagenase and DNAase and then incubated in the same media with 500 rpm lateral shaking at 37 C for 45 min. The single-cell suspension was obtained by passaging the digested tumors through a 70 um filter and washing with PBS. Cells were centrifuged at 350 g for 5 minutes at 4 C and then pellets were resuspended with ACK lysis buffer to lyse red blood cells at room temperature for 10 min and washed with cold PBS. The samples were enriched for immune cells with EasySep Mouse CD45 Positive Selection Kit following the manufacturer's guidelines. The viability of single cells was assessed using Acridine Orange/Propidium Iodide viability staining reagent. Debris-free suspensions of > 70% viability were deemed suitable for the experiments. scRNA-Seq was performed using the Chromium platform (10x Genomics) with the 5' gene expression (5' GEX) V2 kit, with a targeted recovery of 8,000 cells. Briefly, Gel-Bead in Emulsions (GEMs) were generated on the sample chip K in the Chromium X (10x Genomics). Barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup and amplified for 13 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly A-tailing, adapter ligation, and 10x-specific sample indexing following the manufacturer's protocols. Libraries were quantified using TapeStation (Agilent) and QuBit (ThermoFisher) analyses.