Extended Data Figure 6 |. Yeast-displayed deep mutational scanning to map mutations that reduce binding of immunized mouse serum.

(a) Top: Representative plots of nested FACS gating strategy used for all experiments to select for single yeast cells. Bottom: Gating strategy to select for RBD-expressing single cells (FITC-A vs. FSC-A). (b) FACS gating strategy for one of two independent libraries to select cells expressing BA.1 or WH1 RBD mutants with reduced Strep or FLAG antibody binding (cells in blue), as measured by secondary staining with APC-conjugated streptavidin or APC-conjugated anti-FLAG antibody, respectively. Gates were set manually for each sample to capture cells that have a reduced amount of tagged antibody binding for their degree of RBD expression. FACS scatter plots were qualitatively similar between the two libraries. The mouse identifier (#1–4), DMS target library (WH1 or BA.1), and antibody tag (Strep or FLAG) are indicated above each plot. (c) Mutation (left)- and site (right)-level correlations of escape scores between two independent biological replicate libraries.