GSM8743457: Gene expression: chip 2 pool 16; Homo sapiens; RNA-Seq
Bioposied or resected tumors were cut into small pieces with scissors and enzymatically digested by shaking for 1 hour at 37 degrees in RPMI with 10% fetal bovine serum, 0.125 mg/ml collagenase I, 0.125 mg/ml collagenase IV, 0.05 mg/ml DNase I, and 0.05 mg/ml elastase. After digestion, tissue was forced through a cell strainer and lymphocytes were isolated by collecting the interface of a 44%/67% Percoll gradient. Cells were washed with 2% FBS in PBS and viably cryopreserved in FBS with 10% DMSO.  Cryopreserved cells were thawed simultaneously to avoid batch effects, and T cells from each patient were isolated in a single experiment by fluorescence-activated cell sorting by isolating live CD45+CD3+ cells from each sample. Cells with compatible hashing barcodes were pooled together for single-cell RNA-sequencing. From each pool, gene expression (GEX), CITE-seq, and T cell receptor (TCR) libraries were generated with the 10x Genomcs GEM-X reagents according to the manufacturer's protocols (10x Genomics User Guide CG000734). For TCR amplification, primers were also included to amplify γ/δ TCRs. Primers were for TCRγ (outer: TGTGTCGTTAGTCTTCATGGTGTTCC, inner: GATCCCAGAATCGTGTTGCTC), and for TCRδ (outer: AGCTTGACAGCATTGTACTTCC, inner: TCCTTCACCAGACAAGCGAC).