Cd27 percp cy5

PBMCs were thawed and treated with 100 μg/ml DNase1 for 15 min before staining. Tetramers were stained alongside fixable viability dye-eFluor 780 (Thermofisher), CD3-Pacific Blue, CD19-FITC, CD27-PerCP Cy5.5, and IgD-BV605 antibodies (Biolegend or BD) for cell sorting stains. Spike-reactive B cells were sorted based on double positive tetramer staining and submitted to the Princess Margaret Genomics Centre (Toronto, ON) for single cell RNA sequencing. Single-cell library preparation and sequencing was performed using a 10× Genomics Chromium controller to prepare libraries for gene expression (GEX) (Single Cell 5′ R2-only Chemistry) and B cell receptor (BCR) (Single Cell V(D)J R2-only). Libraries for samples were sequenced on an Illumina-2500 for single-cell gene expressions (5000 cells, 50 K reads per cell) and the single-cell BCR sequences (5000 cells, 5 K reads per cell). Our data consisted of single-cell GEX and BCR sequencing data for 3 S-reactive enriched samples and computational antibody discovery was based on the single-cell RNAseq gene expression and BCR sequencing data. Due to the low numbers of purified S-protein reactive cells, purified T cells were added as carriers to facilitate processing.