GSM2442414: P14_r2; Mus musculus; RNA-Seq
Cell suspensions were prepared from tumors. Tumors were dissected, transferred to RPMI medium supplemented with TL Liberase Cocktail (100ug/mL, Roche) and incubated at 37°C for 15 minutes. Then, additional Liberase was added, incubating with mixing for an additional 10 minutes. Tissue suspensions were passed through 70-micron strainers and washed with PBS/2% FBS. For sorting of adoptively transferred OT-I and P14 cells, CD8+ T cells were pre-enriched from tumors using the Dynabeads FlowComp Mouse CD8 selection kit (Thermo Fisher). To ensure adequate cell yield, samples from multiple mice belonging to the same group were pooled prior to enrichment. Enriched CD8+ T cells were then stained with anti-CD8a (53-6.7), anti-CD45.1 (A20), anti-Thy1.1 (OX-7), anti-PD-1 (29F.1A12), anti-Tim-3 (RMT3-23) and Fixable Viability Dye eFluor780. Tumor infiltrating lymphocytes were isolated as live CD8+CD45.1+ (OT-I) or CD8+Thy1.1+ (P14) on BD FACSARIA III or FUSION sorters. Total RNA was extracted from 5,000-30,000 sorted CD8+ T cells using the RNeasy micro kit or the miRNeasy micro kit. RNA quality was evaluated with the Bioanalyzer RNA pico kit (Agilent Technologies). Libraries were prepared from polyA+ RNA using the Whole Transcriptome Amplification Sequencing Technology SEQR kit (Sigma-Aldrich)
GEO Accession
GSM2442414