title: RP DMS Lib Replicate 3, vehicle, bin B
design: A previously described pcDNA5 vector containing rhodopsin, an N-terminal hemagglutinin (HA) tag, an internal ribosome entry site-dasher GFP cassette, and a Bxb1 recombination site in place of the promoter(18) was used to generate a molecular library of barcoded retinopathy variants. We first installed a randomized ten nucleotide barcode region upstream of the Bxb1 recombination site using nicking mutagenesis. A plasmid preparation containing a mixed population of barcoded vectors was used as a template for 123 individual site-directed mutagenesis reactions to generate a library of retinopathy variants that were found in either the Uniprot Database, the Human Gene Mutation Database, and/ or the Leiden Open Variation Database. Individual clones from each reaction were generated using the GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific, Waltham, MA) and sequenced to confirm the sequence of each mutated open reading frame and to determine its corresponding 10 base barcode sequence. Plasmids encoding individual variants were pooled and electroporated into electro-competent NEB10 cells (New England Biolabs, Ipswitch, MA), which were then grown in liquid culture overnight and purified using the ZymoPure endotoxin-free midiprep kit (Zymo Research, Irvine, CA). The Bxb1 recombinase expression vector (pCAG-NLS-HA Bxb1) was provided by Douglas Fowler. A pool of recombinant stable cells expressing individual retinopathy variants was generated using a previously described stable HEK293T cell line containing a genomic Tet-Bxb1-BFP landing pad.(17) Recombinant cells were generated and isolated as was previously described.(18) Briefly, cells grown in 10 cm dishes in complete Dulbeccos modified Eagle medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Corning, Corning, NY) and penicillin (100 U/ml)/streptomycin (100 g/ml) (complete media) were co-transfected with our library of retinopathy variants and the Bxb1 recombinase expression vector using Fugene 6 (Promega, Madison, WI). Doxycycline (2 g/mL) was added one day after transfection and the cells were grown at 33 C for the following 4 days. On the 4th day, cells were sorted using the BD FACS Aria II (BD Biosciences, Franklin Lakes, NJ) to isolate GFP positive/ BFP negative cells that had undergone recombination. These cells were grown in 10 cm dishes with complete media supplemented with doxycycline (2 g/mL) for up to seven days. Where indicated, cells were incubated with 5 M 9-cis-retinal for 16 hours prior to sorting. Rhodopsin expressed at the plasma membrane of recombinant cells was labeled with a DyLight 550conjugated anti-HA antibody (ThermoFisher, Waltham, MA). Labelled cells were then fractionated into quartiles according to surface immunostaining intensity using a FACS Aria IIu fluorescence activated cell sorter (BD Biosciences, Franklin Lakes, NJ). At least 2 million cells from each fraction were isolated to ensure exhaustive sampling. Fractionated sub-populations were expanded in 10 cm culture dishes prior to harvesting and freezing 10-20 million cells per quartile fraction for the downstream genetic analysis. To track the expression of individual retinopathy variants, we first extracted the genomic DNA (gDNA) from each cellular fraction using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich, St. Louis, MO). A previously described semi-nested polymerase chain reaction (PCR) technique(17) was then used to selectively amplify the barcoded region of the recombined plasmids within the gDNA. Briefly, an initial PCR reaction was used to first amplify the region of interest from the gDNA. The product of this reaction was then used as a template for a second PCR reaction that amplified the barcoded region while installing indexed Illumina adapter sequences. Amplicons were gel-purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). The purity of each sequencing library was confirmed using an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA). Libraries were sequenced using a NextSeq 500 Mid Output 150-cycle kit (Illumina, San Diego, CA) at an average depth of ~2 million reads per quartile. These fastq files corresponds to reads obtained from biological replicate 3, FACS quartile B (of four fractions A, B, C, and D), with no treatment of 5 micromolar 9-cis-retinal added to cells, forward (R1) and reverse (R2) paired-end reads.