To conduct the cell surface expression multiplexed assay of KCNE1 , we used an HA epitope in KCNE1, constructed a comprehensively mutated barcoded plasmid pool, and integrated the library into the LP-KCNQ1 cell line. Cells were stained with a fluorophore-conjugated anti-HA antibody, sorted cells into 4 bins by surface KCNE1 levels and each pool was deep sequenced. Variant abundance in each sequenced pool was used to calculate a trafficking score for each variant. The diagram shows three example variant plasmids, each denoted by a different color. The three example variants have different levels of KCNE1-HA surface expression (green: loss-of-trafficking, gray: normal trafficking, yellow: high trafficking), resulting in different patterns of sequencing results in each of the 4 sorted cell pools.
