title: RHO S297R DMS Library Replicate 2, 27 degrees, Plus 9-cis-retinal treatment, Bin B
design: Experimental DMS measurements were collected for this library as previously described (Wesley D Penn et al., 2020). Briefly, a genetically modified HEK293T cell line bearing a single genomic recombination site was used to create a pool of recombinant cells in which each cell inducibly expresses one double mutant from the plasmid pool. Recombined cells were isolated by fluorescence activated cell sorting (FACS) based on their characteristic bicistronic GFP expression. Surface immunostaining was then used to mark the surface rhodopsin using a Dylight-550-labeled anti-HA antibody (Thermo Scientific, Waltham, MA) prior to the fractionation of the cellular population into four quartiles by FACS according to the relative surface immunostaining intensities of the expressed double mutants. Cellular isolates were then grown to confluency and harvested prior to the extraction of their genomic DNA using a X kit (Sigma Aldrich, St. Louis, MO). A nested PCR reaction was then used to amplify the recombined rhodopsin genes and incorporate them into sequencing libraries. The relative abundance of each variant within each isolate was then used to approximate the mean fluorescence intensity of cells expressing each double mutant. Mutant scores were normalized relative to the mean fluorescence intensity of an equivalent set of cells expressing WT rhodopsin. To correct the baseline of these scores, we subtracted the average DMS score of the internal nonsense variants from the DMS scores of the missense variants. This sample corresponds to the second quartile (bin B) from the second replicate experiment on the S297R background library conducted at 27 degrees in the presence of 9 cis retinal.