title: GSM6885356: patient12_T0_CD45; Homo sapiens; RNA-Seq
library_construction_protocol: 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. 40'000 CD45+ cells and 40'000 total live cells were sorted on a MoFlo Astrios (Beckman Coulter) and collected in separated 0.2mL PCR tubes containing 10ml in PBS + 0.04% BSA + 0.1% RNasin. After sorting, cells were manually counted with hemocytometer and viability was assessed using Trypan blue exclusion. Ex vivo CD45 cells from tumor were resuspended at a density of 600-1200 cells/ml when possible with a viability of >90% and subjected to a 10x Chromium instrument for single-cell analysis. Single-cell RNA libraries were generated using the Chromium Next GEM Single Cell 5' Library and Gel beads kit v1.1 for the CD45-sorted population or using the Chromium Next GEM Single Cell 3' Library and Gel beads kit v3.1 for the live-sorted cells, according to the manufacturer's instructions. For each sample, 15'000 cells were loaded into the Chromium machine when possible (i.e. the total of sorted cells if <15'000), encapsulated and barcoded following the manual (CG000388 for 3' or CG000207 for 5' technology). After encapsulation and reverse transcription, 14 PCR cycles were used to amplify cDNA. All library construction steps were performed according to the manufacturer's protocol. For the FACS sorting of post-ACT biopsies, depending of the percentage of CD45+ fraction in the sample, two sorting strategies were adopted. If the CD45+ fraction was inferior to 75% of total viable cells, 40'000 CD45+ and 40'000 CD45- viable cells were sorted in two separated tubes. If the CD45+ fraction was superior to 75% of total viable cells, 40'000 total viable cells were collected in one tube.  All cells were sorted on a MoFlo Astrios (Beckman Coulter) and collected in separated 0.2mL PCR tubes containing 10l in PBS + 0.04% BSA + 0.1% RNasin. After sort, cells were manually counted with hemacytometer and viability was assessed using Trypan blue exclusion. If CD45+ and CD45- were sorted separately, cells were mixed with a ratio of 70% CD45+ and 30% CD45-. All cells were resuspended at a density of 600-1200 cells/mL if possible with a viability >90% and subjected to a 10x Chromium instrument for single-cell analysis. Only the CD45+ compartment was analyzed in this study.