GSM8606573: P14, mouse PD1 (GEX); Mus musculus; OTHER
Tumors were harvested from mice, minced with razors and digested with 1 mg/mL collagenase type I and 1 mg/mL soybean trypsin inhibitor in 1x HBSS at 37 ºC for 1 h, filtered through 70-μm cell strainer (BD), and resuspended in 1x PBS to prepare single-cell suspensions. The tumor single-cell suspension samples were subjected to percoll gradient centrifugation at 800 g for 10 min, after which CD8+ T cells were isolated using a CD8 beads selection kit (Biolegend).　Intratumoral CD8+ T cells were stained with Zombie Aqua Fixable Viability Dye (Biolegend) in 1x PBS on ice for 15 min, and subsequently with BV785 anti-moCD8, PE anti-Thy1.1 and FITC anti-TCRVα2 (P14 TCR) in 1x PBS containing 2% FBS on ice for 30 min. Live, CD8+, Thy1.1+, and TCRVα2+ cells were sorted on FACSAria into different tubes containing RPMI-1640 with 5% FBS. The sorted cell samples were distributed into two groups; cell samples isolated from mice that received moPD1-expressing P14 cells, or cell samples isolated from mice that received moPD1huICD-expressing P14 cells. Each cell sample in the same group was hash-tagged with a unique TotalSeq anti-mouse MHC-I antibody (Biolegend) on ice for 30 min, washed with 1x PBS containing 2% FBS. After the hash-tagging, the cell samples in the same group were pooled into one tube and diluted in 1x PBS containing 2% FBS. Both the gene expression (GEX) library and the hash-tag library of each group were prepared using a Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) with Feature Barcode Technology for Cell Surface Protein (10x Genomics) following manufacturer's instructions. The prepared libraries were quantified and quality checked using TapeStation (Agilent).