GSM2442420: OTI-Ctrl_r3; 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 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+ 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). Poly(A)-selected RNA was amplified using the Smart-seq2 (Clontech) protocol with minor modifications. Briefly, purified RNA was reverse-transcribed using Superscript II, Oligo dT30 VN primers and template switching primers. A pre-amplification step of 8 PCR cycles was performed using the Kapa HiFi Hotstart kit (Kapa Biosystems). The PCR product was purified using AMPureXP beads (Beckman Coulter) and 1 ng was further used for library preparation using the Nextera XT LibraryPrep kit (Illumina). Tagmented DNA was amplified with a 12-cycle PCR reaction and again purified with AMPureXP beads.
GEO Accession
GSM2442420