title: GSM9662921: R3, responder 3; Homo sapiens; RNA-Seq
library_construction_protocol: Cryopreserved tissue was thawed at 37 °C and immediately washed and placed in solution of RPMI and 5% fetal bovine serum (FBS) with the addition of and disassociated using DNAse 1 and Collagenase P at 37 °C for 40 minutes. Digestion was stopped with 100 mM EDTA. Cells were filtered through a 70-µm pore size filter to obtain a single-cell suspension and washed with solution of RPMI + 5% FBS + 2 mM EDTA. Cells were spun down and resuspended in phosphate-buffered saline (PBS) to count. Cells were then stained with Live/Dead Fixable Aqua (Thermo Fisher Scientific) for 20 minutes. Cells were then washed and stained with CD45 (BV510; BD), CD3 (APC; BD), and EpCAM (BV605; BioLegend) with the addition of human Fc block and Brilliant buffer plus (BD). CD45+ and CD3+ cells (T cells) and CD45− and EpCAM+ (epithelial cells) were sorted on the BD FACSAria II and collected into tubes with PBS + 1% bovine serum albumin. Cells were then stained with trypan blue and manually counted with a hemocytometer and resuspended with the desired concentration of 1000 cells/µL. Any cell population with <10 000 cells were combined with another population. Cells were then sent to the Johns Hopkins Sidney Kimmell Comprehensive Cancer Center Experimental and Computational Genetics Core for library preparation and sequencing.  output. The single-cell 5′ V(D)J and 5′ digital gene expression kits (10x Genomics) were used with an emulsion-based protocol provided by the manufacturer. Cells and barcoded gel beads were partitioned into nanoliter-scale droplets using the 10x Genomics Chromium platform to partition up to 10 000 cells per sample (when possible) followed by RNA capture and cell-barcoded complementary DNA synthesis using the manufacturer's standard protocols. Libraries were generated and sequenced on an Illumina NovaSeq instrument using 2 × 150-bp paired end sequencing. The 5′ VDJ libraries were sequenced to a depth of ∼5000 reads per cell and the 5′ differential gene expression libraries were sequenced to a target depth of ∼50 000 reads per cell. Cell Ranger (v9.0; 10x Genomics) was used to align reads to reference (GRCh38-3.0.0) and generate feature-barcodematrices from the Chromium scRNAseq