Isolation of SARS-CoV-2 RBD-specific memory B cells

Probe-specific single B cells were sorted as previously described [27 (link)]. Briefly, one tube of PBMCs was thawed in water at 37 °C and resuspended in a prewarmed RPMI-1640 (Corning, Corning, NY, USA) containing 10% fetal bovine serum (Sera Pro, Gremlingen, BW, Germany) and 50 IU/mL benzonase (Sigma, St. Louis, MO, USA). After washing with PBS, the PBMCs were stained with a LIVE/DEAD dye (Invitrogen, Carlsbad, CA, USA) on ice for 20 min. After washing, the PBMCs were stained with an antibody cocktail containing anti-CD3-Pacific Blue, anti-CD8-Pacific Blue, anti-CD19-BV510, anti-IgM-PercpCy5.5, anti-IgG-FITC, anti-CD27-APC-Cy7, anti-PD-1-PECy7, anti-CXCR5-APC-R700, anti-CXCR3-PECy5, anti-CD45RA-BV650, anti-CD4-BV605 (above antibodies are all from BD Biosciences), anti-CD14-eflur450 (ebioscience, USA), anti-CD20-ECD (Beckman Coulter, Bria, CA, USA) and RBD-PE. After filtering through a cell strainer (Corning, Corning, NY, USA), the PBMCs were sorted on BD FACS Aria SORP. RBD-specific single memory B cells were gated as CD3⁻, CD8⁻, DAPI⁻, CD14⁻, CD19⁺, CD20⁺, CD27⁺, IgM⁻, IgG⁺, RBD-PE⁺, and were sorted into a 96-well PCR plate containing cell lysis buffer. Then, the PCR plate was frozen immediately at −80 °C and stored overnight.