CFTR DMS1 Library Replicate 3, DMSO treated, Bin C
DMS studies were carried out using a previously described plasmid library containing 232 CF variants that are encoded in a CFTR cDNA containing a triple hemagglutinin (HA) epitope tag within the fourth extra-cellular loop (MSD-2 domain). This library also features an upstream Bxb1 recombination site to facilitate stable recombination and a downstream internal ribosome entry site (IRES)-GFP cassette, which serves as an expression reporter. This variant library was prepared via electroporation of electrocompetent NEB10 cells (New England Biolabs, Ipswich, MA, USA) and grown in LB broth overnight prior to purification using the ZymoPure endotoxin-free midiprep kit (Zymo Research, Irvine, CA). Western blot and Ussing chamber measurements were carried out using an untagged CFTR expression construct in a pcDNA5 backbone that was generously provided by Eric Sorcher and Jeong Hong (Emory University, Atlanta, GA, USA). The V1240G and Y1032C variants were generated in this backbone by site-directed mutagenesis using the Q5 DNA polymerase and the KLD enzyme kit (New England Biolabs, Ipswich, MA, USA). The Bxb1 recombinase expression vector (pCAG-NLS-HA Bxb-1) that was used to generate recombinant cellular libraries was generously provided by Douglas Fowler (University of Washington, Seattle, WA, USA). The sequences of all plasmids used for these experiments were validated by whole plasmid sequencing (Plasmidsaurus Inc., Watterson Park, KY, USA). Deep mutational scanning (DMS) experiments were carried out as previously described. Briefly, a genetically-modified HEK293T cell line bearing a genomic attP recombination site was used to prepare a pool of recombinant stable cells in which each cell inducibly expresses one of 232 known CF variants. Cells were grown at 37C in an incubator containing 5% CO2 in Dulbeccos Modified Eagles Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mix (Gibco, Grand Island, NY, USA). Recombination was initiated by co-transfecting cells with a Bxb1 recombinase expression vector and a pooled CF variant library using Fugene 6 (Promega Corp., Madison, WI, USA). Cells were incubated at 33C in an incubator containing 5% CO2 in Dulbeccos Modified Eagles Medium (DMEM) with 10% fetal bovine serum, 1% penicillin/streptomycin for 3 days post-transfection. Expression of CF variants was then initiated 24 hours post-transfection by adding 2 g/ mL doxycycline to the growth medium. A Bigfoot Spectral Cell Sorter (Thermo Scientific, Waltham, MA, USA) was used to isolate recombinant cells expressing individual CF variants based on their characteristic gain in IRES-GFP expression via fluorescence activated cell sorting. Recombinant cellular libraries were then expanded into 15-cm cell culture dishes and treated with the indicated concentrations of small molecules for 16 hours prior to harvesting with 0.25% Trypsin-EDTA (Gibco, Grand Island, NY, USA). Surface CFTR proteins on treated cellular libraries expressing CF variants were then marked using a DyLight 550-conjuaged with anti-HA antibody prior to fractionation of each treated library into four even quartiles ( 1 million cells/ fraction) according to relative surface immunostaining of expressed CF variants using a Bigfoot Spectral Cell Sorter (Thermo Scientific, Waltham, MA, USA). Each cellular isolate was then expanded in a 10 cm dish prior to harvesting and flash-freezing. To quantify the relative abundance of each CF variant within each cellular isolate, we first extracted the genomic DNA (gDNA) from the frozen cell pellets using the DNEasy extraction kit (Qiagen Sciences Inc., Germantown, MD, USA). A previously described nested PCR approach was then used to create DNA amplicons bearing the region of the recombinant gDNA that contains unique molecular identifiers for the expressed CF variants. Amplicons generated for each cellular sub-population were then deeply sequenced (>7 million reads per amplicon) using an Cloudbreak Freestyle cell on an Aviti24 DNA sequencing system (Element Biosciences, San Diego, CA, USA).
SUB16196587
Rep3_CFTR_VehicleControl_BinC