TCR library for sample MC_505B
Biopsies were cut into pieces less than 1mm in diameter with dissecting scissors and placed into RPMI media (ThermoFisher 11835055) with 1 mg/mL of protease from Bacillus lecheniformis (MilliporeSigma P5380), 5 mM CaCl2, and 0.1 mg/ml DNAse I (MilliporeSigma DN25). Tissue dissociation mix was incubated at 4C for 40 minutes, under constant rotation. Tissue was triturated every 10 minutes with a P1000 pipette. The dissociation reaction was stopped by adding human AB serum (MilliporeSigma H4522) to a final volume of 10%. The dissociated mix was filtered through a 70 µM filter to remove clumps, then centrifuged for 10 minutes at 350g. The cell pellet was resuspended in RPMI media, and cells were counted in a hemocytometer. Cells were then cryopreserved in a 1:1 solution of RPMI : CryoStor CS10 (BioLife 210102) for a final DMSO concentration of 5%. After thawing a vial of cells, single-cell suspensions were brought up in phenol-free RPMI with 2% (v/v) human AB serum and incubated on ice for 30 min with the following antibodies: CD66b-FITC (1:100, BioLegend, 305104), EpCAM-PE (1:100, BioLegend, 324206), CD45-APC (1:150, BioLegend, 304012), CD3 PerCP-Cy5.5 (1:150, BioLegend, 300328) and CD235a PE-Cy7 (1:150, BioLegend, 349112). Cells were washed once and resuspended in phenol-free RPMI with 2% (v/v) human AB serum containing DAPI (Thermo Fisher Scientific, 62248). Single-color controls were performed with these same antibodies using BD CompBeads (BD Biosciences, 552843). Live (that is, DAPI−), singlet, CD66b−CD235a−EpCAM−CD45+ cells were sorted into phenol-free RPMI with 2% (v/v) human AB serum. All sorting was performed on Sony SH800 or MA900 cell sorters. At least 20,000 immune cells were sorted from each patient. Isolated CD45+ cells were centrifuged and resuspended at a concentration of 800–1,200 cells per microliter in RPMI with 2% (v/v) human AB serum and loaded onto a 10X Chromium instrument with the following kit: Chromium Single Cell 5' V1 (10X Genomics product PN-1000006). The targeted recovery was 4,000 cells. The manufacturer's protocol was followed to generate sequence-ready libraries. TCR amplification/library generation was performed according to manufacturer directions (10X Genomics kit PN-1000005 and PN-1000016). Libraries were sequenced on an Illumina NextSeq. 10X Genomics Cell Ranger (version 3.1.0) was used to demultiplex reads.