title: GSM8368632: Tumor_raw_TCR_P7; Homo sapiens; OTHER
library_construction_protocol: Tumor samples were transported in MACS® Tissue Storage Solution stored at 4°C, rinsed with PBS, minced and incubated in a rotation shaker for 40 minutes at 37°C in Collagenase IV 0.25mg/ml, Collagenase D 200U/ml and DNAse I 0.1mg.ml (all Sigma). Cell suspensions were then aspirated through a 18G needle ten times and strained through a 70-micron mesh prior to RBC lysis. Dead cells were removed using an EasySep Dead Cell Removal (Annexin V) Kit. Cell suspensions were sorted into CD45+ and CD45- cells using the EasySepTM Human CD45 Depletion Kit per kit instructions. Single-cell RNA-seq (scRNA-seq) was performed on tumor samples using the Chromium platform (10x Genomics, Pleasanton, CA) utilizing both the 3′ and 5' gene expression kits. Approximately 6000 CD45+ and CD45- cells were loaded into each channel of the 10x Chromium controller, following the manufacturer-supplied protocol. For 5' gene expression samples, BCR and TCR CDR3 sequences were enriched using the human V(D)J B/T cell enrichment. 10x libraries were constructed using the 10x supplied protocol and sequenced at the Mount Sinai Genomics Core Facility. Gel-bead in emulsions (GEMs) were generated on the sample chip in the Chromium controller. Barcoded cDNA was extracted from the GEMs using Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly-A-tailing, adaptor ligation, and 10X-specific sample indexing following the manufacturer's protocol. Libraries were quantified using Bioanalyzer (Agilent) and QuBit (Thermofisher) analysis and then sequenced in paired-end mode on a HiSeq 2500 instrument (Illumina, San Diego, CA).