Isolation and Characterization of SARS-CoV-2 Specific B Cells

PBMCs were enriched for B cells by negative selection using a pan B cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were incubated in FACS buffer (1 X Phosphate-buffered Saline (PBS), 2% calf serum, 1 mM EDTA) with the following anti-human antibodies: anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41), anti-CD8-APC-eFluro 780 (Invitrogen, 47-0086-42), anti-CD16-APC-eFluro 780 (Invitrogen, 47-0168-41), anti-CD14-APC-eFluro 780 (Invitrogen, 47-0149-42), as well as Zombie NIR (BioLegend, 423105), and fluorophore-labeled RBD and Ovalbumin for 30 minutes on ice^{27 (link)}. Single CD3⁻CD8⁻CD16⁻CD20⁺Ova⁻RBD-PE⁺RBD-AF647⁺ B cells were sorted into individual wells of a 96-well plates containing 4 μl of lysis buffer (0.5 X PBS, 10mM DTT, 3000 units/mL RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III (Becton Dickinson). The sorted cells were frozen on dry ice, and then stored at −80°C or immediately used for subsequent RNA reverse transcription. Although cells were not stained for IgG expression, they are memory B cells based on the fact that they are CD20+ (a marker absent in plasmablasts) and they express IgG (since antibodies were amplified from these cells using IgG-specific primers).