GSM9657545: Tet2fl/fl CD4+ TILs, biol rep 2; Mus musculus; RNA-Seq
Tumor-infiltrating leukocytes were isolated from liver metastatic tumors by enzymatic digestion with collagenase A (0.75 mg/ml) and DNase I (50 µg/ml) followed by density gradient centrifugation using Histopaque-1083. CD45+ cells were enriched by MACS separation. CD4+ and CD8+ TILs were sorted by flow cytometry using anti-CD45, anti-CD3ε, anti-CD4, and anti-CD8α antibodies on a BD FACSAria III cell sorter. Total RNA was extracted from sorted cells using the RNeasy Micro Kit (Qiagen). RNA integrity was assessed using the Agilent 2100 Bioanalyzer. Samples with RIN > 7 were used for library preparation with the SMARTer Stranded Total RNA-seq Kit v2 - Pico Input Mammalian (Takara Bio, Cat#634413) according to the manufacturer's protocol.