GSM7962425
Baseline tumors, either frozen pieces were thawed in RPMI + 20% FBS on the day of the assay or fresh tissue was directly chopped upon reception. Tissue was dissociated in RPMI + 2% Gelatin (Sigma-Aldrich) + 200 IU·mL-1 Collagenase I (ThermoFisher Scientific) + 400 IU·mL-1 Collagenase IV (ThermoFisher Scientific) + 5 IU·mL-1 Deoxyribonuclease I (Sigma-Aldrich) + 0.1% RNasin Plus RNase Inhibitor (Promega) for 15-30 min at 37°C. Digested cells were filtered using a 70 μm strainer and resuspended in PBS + 1% Gelatin + 0.1% RNasin. Cells were manually counted then stained with 50 µM·mL-1 of Calcein AM (Thermo Fisher Scientific) and FcR blocked (Miltenyi Biotec) for 15 min at RT. Surface staining with anti-CD45 (BioLegend) was performed for 20 min at 4°C. Cells were resuspended in PBS + 0.04% BSA (Sigma-Aldrich) and DAPI (Invitrogen) staining was performed. 40'000 CD45+ cells were sorted on a MoFlo AstriosEQ (Beckman Coulter) and collected in 0.2mL PCR tubes containing 10 μL of PBS + 0.04% BSA + 0.1% Rnasin. After sorting, cells were manually counted with a hemocytometer and viability was assessed using Trypan blue exclusion.  Ex vivo CD45+ cells from tumors were resuspended at a density of 600-1200 cells µL-1 with a viability of >90% and subjected to a 10X Chromium instrument for single-cell analysis. The standard protocol of 10X Genomics was followed and the reagents for the Chromium Single Cell 5' Library and V(D)J library (v1.0 or v1.1 Chemistry) were used. 15'000 cells were loaded per sample when possible (or the total number of sorted cells if < 15'000 cells). Using a microfluidic technology, single-cell were captured and lysed, mRNA was reverse transcribed to barcoded cDNA using the provided reagents (10X Genomics). 14 PCR cycles were used to amplify cDNA and the final material was divided into two fractions: first fraction was target-enriched for TCRs and V(D)J library was obtained according to manufacturer protocol (10X Genomics). The second fraction was processed for 5' gene expression library following the manufacturer's instruction (10X Genomics). Complementary DNA and library quality were examined on a Fragment Analyzer (Agilent) and quantification was performed with the Qubit HS dsDNA assay kit (Invitrogen). Barcoded V(D)J libraries and GEX libraries were pooled and sequenced by an Illumina HiSeq 4000 sequencer. scTCR-seq