title: Epitope mapping using charged-scanning mutagenesis
design: Charged scanning mutagenesis library of SARS-CoV-2 RBD was displayed on the yeast surface as a novel approach to identify the epitopes targeted by the antibodies present in the polyclonal serum samples. WA.1 RBD (332-532) was used as the template for library construction and all RBD residues with solvent accessibility >15% were individually mutated to negatively charged Aspartate, with the exception of negatively charged residues, which were mutated to the positively charged Arginine residue. The mutants were linked to 6N barcode of known sequence. The generated RBD library was displayed on the yeast surface and subjected to binding with a saturating concentration of the polyclonal serum sample as determined by sera titration with the WT RBD displayed on the surface of the yeast cells. Binding was detected using fluorescently labelled antibodies, and the yeast cell populations were sorted into different bins through Fluorescence Activated Cell Sorting (FACS). Yeast populations within these bins were then subjected to deep sequencing.