Sequencing SARS-CoV-2 Spike-Specific Memory B Cells

S2P spike trimer-specific memory B cells were isolated and sequenced using the protocol previously described by Liu et al.²¹ (link)^,27 (link) In brief, peripheral blood mononuclear cells (PBMCs) from patient 12 were stained with LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Invitrogen) at room temperature for 20 min, washed with RPMI-1640 complete medium, and incubated with 10 μg/mL B.1.351 S2P spike trimer at 4 °C for 45 min. Cells were then washed and incubated with a cocktail of flow cytometry and hashtag antibodies, containing CD3 PE-CF594 (BD Biosciences), CD19 PE-Cy7 (Biolegend), CD20 APC-Cy7 (Biolegend), IgM V450 (BD Biosciences), CD27 PerCP-Cy5.5 (BD Biosciences), anti-His PE (Biolegend), and human Hashtag 3 (Biolegend) at 4 °C for 1 h. Cells were then washed again, resuspended in RPMI-1640 complete medium, and sorted for S2P spike trimer-specific memory B cells (CD3−CD19+CD27+S trimer+ live single lymphocytes). These sorted cells were mixed with PBMCs from the same donor, labeled with Hashtag 1, and loaded to a 10X Chromium chip for the 5′ Single Cell Immune Profiling Assay (10X Genomics) at the Columbia University Human Immune Monitoring Core (HIMC; RRID:SCR_016740). Library prep and quality control were performed according to the manufacturer’s instructions and then sequenced on a NextSeq 500 (Illumina).