GSM5449996: Group C - Patient 1 Late-TCR; Homo sapiens; OTHER
Frozen PBMCs were removed from liquid nitrogen and resuspended in warm RPMI media + 10% FBS (@ 37ºC) by adding 1 mL, 3 mL, 4 mL, 8 mL 16 mL media at 30-60 second intervals. Cells were washed and passed through a 40 um filter, centrifuged @ 300 rcf x 5 min and resuspended in PBS with 50 ul 1% BSA and Biolegend Fc Receptor Blocking Solution (Human TrueStain FcX (Cat. No. 422301) for 20 minutes. Afterwards, another 50 uL of antibody mastermix containing Biolegend TotalSeq-C antibodies for staining (Biolegend TotalSeq™-C0063 anti-human CD45RA Antibody Cat. No. 304163; Biolegend TotalSeq™-C0072 anti-human CD4 Antibody Cat. No. 300567; Biolegend TotalSeq™-C0046 anti-human CD8 Antibody Cat. No. 344753) for final manufacture-recommended concentration of 1.0 ug of antibody in 100 ul of staining buffer for every 1 million cells for a total of 10-15 minutes incubation. After incubation, cells were washed 2x by resuspending in 1.5 mL PBS + 1% RNase-free BSA (MACS BSA Stock Solution No. 130-091-376) and then 1 mL PBS + 0.04% BSA. 10 uL of cell suspension was then taken for counting and appropriate volume of PSA + 0.04% BSA was added (200-500 uL) to aim for final target concentration of 1 million cells/mL). Cells were then loaded into Chromium Next GEM Chip G (10x PN 2000177) to achieve 6000-8000 target cell recovery per sample according to the manufacture's protocol. All steps from cDNA isolation to library preparation were completed according to 10x's manufacturing protocols for the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1. Isolated cDNA was amplified (14 cycles), and then cDNA was allocated for TCR enrichment, feature barcode library generation, and 5' cDNA library generation. cDNA and library quality was evaluated using the high sensitivity DNA kit on the Agilent 2100 Bioanalyzer.