GSM8907774: antiPD1_treatment; Mus musculus; RNA-Seq
Cell surface staining of single-cell suspensions from tumors was performed after two times wash with FACS buffer (PBS supplemented with 2% FBS). All samples were first stained with LIVE/DEAD™ Fixable Blue to exclude dead cells and incubated with Fc receptor blocking antibody (Biolegend, #101320). Then, cells were stained with anti-CD45 (30-F11) and anti-CD3 (17A2). Biological replicates for saline control (n=2) and treated group (n=4) were individually processed. Biological replicates were then pooled together at the single cell suspension stage before sorting with equivalent number of cells from each replicate. The following populations were sorted: P1: CD45+CD3+ (TIL cells), P2: CD45+CD3− (non-TIL immune cells), P3: CD45−CD3− (tumor and stromal cells). P1, P2, and P3 for each sample were then mixed back together at a 9:9:2 ratio, respectively. 20,000 cells from each treatment condition were loaded onto the 10x Genomics Chromium System. Library preparation was performed with 10x Genomics reagents according to the manufacturer's instructions by the Yale Center for Genome Analysis (YCGA). Libraries were then sequenced with an Illumina NovaSeq 6000 at the YCGA.