Mutliplexed single cell sequencing of TCR genes for bioreplicate 2.
PBMCs from 5 healthy donors were quickly thawed and placed in pre-warmed xVIVO15 cell culture medium (Lonza) supplemented with 5% heat-inactivated FBS. To reduce cell clumping, PBMCs were incubated in xVIVO15 containing 50 U per mL of benzonase nuclease (Sigma-Aldrich) for 15 minutes at 37 degrees and filtered using a 70 µm cell strainer. Washed and nuclease treated cells were seeded in a 96 well cell culture plate at a concentration of 2.5 x 106 per mL. Peptide stimulations were performed using the CEFX Ultra SuperStim Pool (JPT Peptide Technologies, Product Code PM-CEFX-1) at a final concentration of 1.25 µg per mL per peptide for 22 hours at 37 degrees and 5% CO2. Recombinant anti-CD28 and anti-CD49d antibodies (BioLegend) were added at a final concentration of 5 µg per mL and 0.625 µg per mL, respectively, to provide co-stimulation for peptide reactive T-cells. Separately mock-stimulated cells were treated with anti-CD28 and anti-CD49d antibodies at the same concentration. Peptide responsive T-cells were detected by the expression of the surface activation markers PD-L1, OX40, and CD137 via flow cytometry. Following the stimulation, peptide treated and mock-stimulated cells were washed in cell staining buffer (PBS + 2mM EDTA + 2% FBS) to end the stimulation. Fc receptor blocking was performed using a 1 to 50 dilution of Human TruStain FcX (Biolegend) in cell staining buffer for 10 minutes at 4 degrees. Cell viability staining was performed using a 1 to 500 dilution of Zombie Yellow Fixable Viability Dye (BioLegend) prepared in PBS for 30 minutes at 4 degrees. Surface staining was performed using 1 to 100 dilutions of BV421 conjugated anti-CD3, FITC conjugated anti-CD4, BV786 conjugated anti-PD-L1, PE conjugated anti-OX40, and APC conjugated anti-CD137 (BioLegend) for 25 minutes at 4 degrees in cell staining buffer. Following cell staining, antigen reactive and non-reactive T-cells were identified using a BD FACSAria II cell sorter and collected in cRPMI medium (100 U per mL penicillin-streptomycin + 2 mM L-glutamine + 10 mM HEPES + 0.1 mM non-essential amino acids + 1 mM sodium pyruvate + .05 mM 2-Mercaptoethanol) supplemented with 20% FBS. Sorted T cell populations were then labeled with 75 uL of TotalSeq oligo conjugated hashing antibody mix, incubated for 30 minutes at 4 degrees with gentle mixing after 15 minutes, and pooled in equal quantities. Staining with the TotalSeq-C Human Universal Cocktail (BioLegend) was then performed according to the manufacturer's instructions. The cells were then resuspended in PBS supplemented with .04% FBS at a final concentration of 500 cells per µL and submitted for single-cell profiling on the Chromium Next GEM instrument. Library preparation was completed for the hashtag oligos, single-cell rna-seq, cite-seq, and TCR-repertoire sequencing following the manufacturer's instructions.