title: scTCR-seq of Homo sapiens- vaccine skin site
design: For analysis of skin-infiltrating immune cells, two 5mm punch biopsies were obtained from one of the two vaccine sites. Skin samples were minced using scalpels, incubated in digestion enzymes (Miltenyi Whole Skin Dissociation kit, utilizing enzyme P and enzymes A+D), incubated at 37C for two hours with agitation, and further dissociated using the Miltenyi gentleMACS (program h_skin_01). The single-cell suspension was then enriched for CD45+ immune cells through immunomagnetic purification (Miltenyi anti-CD45 microbeads). Cells were washed and then resuspended in 1X PBS with 0.04% UltraPure BSA (Invitrogen) for downstream 10x 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.