Dtt and h2o

Fluorescence-activated cell sorting of cryopreserved PBMCs was performed on a BD FACS Melody as previously described (Graham et al., 2021 (link)). Sorting baits (SARS-CoV-2 Spike and RBD) was pre-complexed with the streptavidin fluorophore at a 1:4 molar ratio prior to addition to cells. PBMCs were stained with live/dead (fixable Aqua Dead, Thermofisher), anti-CD3-APC/Cy7 (Biolegend), anti-CD8-APC-Cy7 (Biolegend), anti-CD14-BV510 (Biolegend), anti-CD19-PerCP-Cy5.5 (Biolegend), anti-IgM-PE (Biolegend), anti-IgD-Pacific Blue (Biolegend) and anti-IgG-PeCy7 (BD) and Spike-Alexa488 (Thermofisher Scientific, S32354) and Spike-APC (Thermofisher Scientific, S32362) or RBD-Alexa488 and RBD-APC. Live CD3/CD8^-CD14^-CD19⁺IgM^-IgD^-IgG⁺Spike⁺Spike⁺ or CD3/CD8^-CD14^-CD19⁺IgM^-IgD^-IgG⁺RBD⁺RBD⁺ cells were sorted using a BD FACS Melody into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H₂O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers (Bioline Reagents Ltd) following the manufacturer’s protocol.

PBMCs were purified from rabbit 8315 seven days after the third boost using a Lymphoprep (STEMCELL Technology) density gradient. PBMCs were cryopreserved in FBS plus 10% DMSO. Fluorescence-activated cell sorting of cryopreserved PBMCs was performed. PBMCs were stained with anti-CD3-FITC (Santa Cruz Biotechnology), anti-IgM-PE (Southern Biotech), anti-IgG-PerCP-Cy5.5 (Santa Cruz Biotechnology) and hexahistidine-tagged RVFV-Gn. Cells were washed and anti-HIS-APC (Abcam) was added. CD3^-IgM^-IgG⁺RVFV Gn⁺ cells were sorted into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H₂O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers following the manufacturer’s protocol.