Isolation of SARS-CoV-2 RBD-Specific B Cells

RBD-specific single B cells were sorted as previously described [29 (link)]. In brief, PBMCs from infected individuals were collected and incubated with an antibody cocktail and a His-tagged RBD protein for identification of RBD-specific B cells. The cocktail consisted of the Zombie viability dye (Biolegend), CD19-Percp-Cy5.5, CD3-Pacific Blue, CD14-Pacific Blue, CD56-Pacific Blue, IgM-Pacific Blue, IgD-Pacific Blue, IgG-PE, CD27-PE-Cy7 (BD Biosciences) and the recombinant RBD-His described above. Two consecutive staining steps were conducted: the first one used an antibody and RBD cocktail incubation of 30 min at 4 °C; the second staining involved staining with anti-His-APC and anti-His-FITC antibodies (Abcam) at 4 °C for 30 min to detect the His tag of the RBD. The stained cells were washed and resuspended in PBS containing 2% FBS before being strained through a 70-μm cell mesh filter (BD Biosciences). RBD-specific single B cells were gated as CD19 ⁺CD27 ⁺CD3^-CD14^-CD56^-IgM^-IgD^-IgG ⁺RBD⁺ and sorted into 96-well PCR plates containing 10 μL of RNAase-inhibiting RT–PCR catch buffer (1M Tris-HCl pH 8.0, RNase inhibitor, DEPC-treated water). Plates were then snap-frozen on dry ice and stored at −80 °C until the reverse transcription reaction.