Next Generation Sequencing of bacterial surface-displayed ENAH EVH1 ligand peptides after a FACS titration sort (MassTitr)

We used bacterial surface display and FACS to sort peptide binders to a tetramerized ENAH EVH1 domain at multiple concentrations of ENAH into 4 gates. This experiment was repeated three times. By calculating the distribution of each clone across gates at multiple concentrations, we are able to assess whether a clone exhibited concentration-dependent binding to ENAH EVH1 domain, allowing us to distinguish binders from nonbinders. A multiplexed, barcoded dataset with three replicate sorts of four bins at eight receptor concentrations