GSM6856999
Resected tumors were chopped into 1-2 mm2 pieces and, along with post-infusion biopsies, cryopreserved in 90% human serum + 10% dimethyl sulfoxide (DMSO) and additional pieces were snap frozen for bulk RNA extraction. For single-cell experiments, both frozen and fresh material were used as starting material. PBMCs were isolated from blood collected in EDTA tubes and cryopreserved in 90% human serum + 10% DMSO. The day of the assay, pathofrozen pieces were thawed in RPMI + 10% FBS and chopped in small pieces using a scalpel. Tissue was dissociated in RPMI + 2% Gelatin (#G7041, Sigma-Aldrich) + 200 IU/mL Collagenase I (#17100-017, ThermoFisher Scientific) + 400 IU/mL Collagenase IV (#17104-019, ThermoFisher Scientific) + 5 IU/mL Deoxyribonuclease I (#D4527, Sigma-Aldrich) + 0.1% RNasin Plus RNase Inhibitor (#N2618, Promega) for 15-30min (depending of sample size and consistency) at 37°C and shaken at 160rpm. Digested cells were filtered using a 70 mm strainer and resuspended in PBS + 1% Gelatin + 0.1% RNasin. Cells were manually counted with hematocymeter then stained for viability with 50uM/mL of Calcein AM (#C3099, Thermo Fisher Scientific) and FcR blocked (#130-059-901, Miltenyi Biotec) for 15min at RT. After incubation and washing, cells were stained with CD45-APC (#304012, BioLegend) for 20min at 4°C. After washing, cells were resuspended in PBS + 0.04% BSA (Sigma-Aldrich) + 0.1% RNasin and DAPI staining (Invitrogen) was performed. Total RNA was extracted from snap frozen tissues. Samples were lyzed in TRIzol reagent (Invitrogen) using a tissue lyzer (Qiagen) and the RNA was purified with the RNeasy kit (Qiagen). RNA quality was assessed with a Fragment Analyzer (Agilent) and Nanodrop spectrophotometer (Thermofisher). Quantification was performed with the Qubit HS RNA assay kit (InVitrogen). RNA sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA reagents according to the protocol supplied by the manufacturer and sequenced using HiSeq 4000.