GSM8121407: TIL, ZT7, replicate 1; Mus musculus; RNA-Seq
Tumor tissue was collected and chopped into small pieces. Digestion medium was added (RPMI containing 1mg/mL collagenase IV (Worthington Biochemical Corporation), 40 μg/mL DNase I (Roche 04716728001) and 2% heat-inactivated FCS) and incubated for 30 minutes at 37 °C. Cells were rinsed through a 70 μm cell strainer to obtain single-cell suspensions. To remove debris, Lympholyte®-M (CEDARLANE) was used according to manufacturer's instructions. Enriched tumor infiltrating leukocytes were stained and sorted (CD45+ DRAQ7-) using a Biorad S3 sorter in PBS with 1% BSA. Cellular suspension was loaded on a 10× Genomics Chromium instrument. Single-cell RNA-Seq libraries were prepared using Chromium Single Cell 3′ v3.1 Reagent Kit with dual indexes according to manufacturer's protocol. Library quantification and quality assessment was performed using a Qubit fluorometer (ThermoFisher Scientific) and a Tapestation (DNA High sensitivity chip - Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq 6000 using paired-end 28 × 90 bp as sequencing mode.