title: CFTR DMS LIBRARY, VX-445+VX-661 (elexacaftor-tezacaftor combination) treatment, replicate 1, bin D
design: A pcDNA5 vector containing CFTR without a promoter, a triple hemagglutinin (HA) tag in the fourth extra-cellular loop, an internal ribosome entry site- eGFP cassette, and a Bxb1 recombination site was used to generate a molecular library of barcoded CF 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 WT plasmid was used as a template for 129 individual site-directed mutagenesis reactions to generate a library of pathogenic CF variants that were found in the CFTR2 Database (https://www.cftr2.org/). Individual clones from each reaction were generated using the Zyppy-96 Plasmid Kit (Zymo Research, Irvine, CA) and deep 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 beta 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). Transient expression of individual variants for western blots was carried out using a distinct set of expression vectors encoding the untagged CFTR variant cDNAs in the context of a pCDNA5 expression vector, which were generated by site-directed mutagenesis. The Bxb1 recombinase expression vector pCAG-NLS-HA Bxb1) was kindly provided by Douglas Fowler. A pool of recombinant stable cells expressing individual CF variants was generated using a previously described stable HEK293T cell line containing a genomic Tet-Bxb1-BFP "landing pad." Recombinant cells were generated and isolated as was previously described. Briefly, cells grown in 10 cm dishes in complete media were co-transfected with our library of cystic fibrosis variants and the Bxb1 recombinase expression vectors using Fugene 6 (Promega, Madison, WI). Doxycycline (2 mcg/mL) was added one day after transfection and the cells were grown at 33 deg C for the following 3 days. The cells were then incubated at 37 deg C for 24 hours prior to the isolation of GFP positive/ BFP negative cells that had undergone recombination using a BD FACS Aria II (BD Biosciences, Franklin Lakes, NJ). These cells were grown in 10 cm dishes with complete media supplemented with doxycycline (2 mcg/mL) until confluency when they were divided into 15 cm dishes for the three drug trial replicates. Where indicated, cells were incubated with either DMSO (vehicle), 3 micromolar VX-445, VX-661, or a combination of the two for 16 hours prior to sorting. CFTR expressed at the plasma membrane of recombinant cells was labeled with a DyLight 550-conjugated 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 surface immunostaining of individual CF variants, we first extracted the 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 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. Surface immunostaining levels were estimated from sequencing data using a computational approach described previously. Briefly, low quality reads that were likely to contain more than one error were removed from the analysis. The remaining reads containing one of the 129 barcodes corresponding to a variant of interest were then rarefied to generate subsampled datasets with a uniform number of reads for each sample. This sample contains the sequenced barcodes for the VX-445+VX-661 (elexacaftor-tezacaftor combination) treatment, replicate 1, bin D.