title: scTCR-seq of Homo sapiens- peptide pool stimulated IFN-y+ T cells
design: For isolation of vaccine-reactive T cells from peripheral blood (patients 101, 103, 104, 108), post-vaccine PBMCs were stimulated in vitro with 10 μg ml-1 IMP pools overnight.  Following stimulation PBMCs were enriched for T cell populations. PBMCs were stained with CD4 and CD8 magnetic beads (Miltenyi Biotec), for enrichment via positive selection. Samples were subsequently stained for IFN-γ secreting cells (IFN-γ Secretion Assay, Miltenyi Biotec). Following IFN-γ capture, samples were stained with additional surface antibodies (CD3 (clone UCHT-1; BD Biosciences), CD4 (clone M-T466; Miltenyi Biotec), CD8 (clone BW135,80; Miltenyi Biotec), CD14 (clone M5E2; BD Biosciences), CD19 (clone HIB19; BD Biosciences). Samples were then washed and stained with viability dye (Zombie NIR Biolegend). Immediately after staining, samples were FACS sorted on the basis of viability, negativity for CD14 and CD19, positive expression of CD3, and IFN-γ co-expressed with CD4 or CD8. Cells were sorted into UltraPure 0.04% BSA. Following sorting, cells were centrifuged to concentrate and supernatant removed for downstream 10x scRNAseq and scTCRseq analysis. Cells were loaded onto a 10x Genomics ChromiumTM instrument (10x Genomics) according to the manufacturer's recommendations. The scRNAseq libraries were processed using Chromium Next GEM Single Cell 5' Kit v2, and the scTCRseq libraries were made using Chromium Single Cell Human TCR Amplification Kit (10x Genomics). Quality controls for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The sequencing libraries for scRNAseq and scTCRseq were normalized to 4nM concentration and pooled. The pooled sequencing libraries were sequenced on Illumina NovaSeq S4 300 cycle platform. The sequencing parameters were Read 1 of 26bp, Read 2 of 90bp, Index 1 of 10bp and Index 2 of 10bp.