Deep Receptor Scanning Illumina Counts
A pcDNA5 vector, in which we inserted a GPCR with a hemagglutinin (HA) tag at the n-terminus or c-terminal to the signal peptide in a computational derived manner. The vector also contains an internal ribosome entry site- dasherGFP cassette, and a Bxb1 recombination site was used to generate a molecular library of barcoded GPCRs. We first installed a randomized ten nucleotide unique molecular identified (UMI) upstream of the Bxb1 recombination site using nicking mutagenesis in place of the CMV promoter. Individual plasmids were prepared by GenScript and verified using nanopore technology. The individual plasmids were then pooled together in an equimolar ratio. The plasmid pool was then electroporated into electro-competent NEB10b 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 kindly provided by Douglas Fowler. CFTR variant DNA constructs were stably recombined in modified HEK293T cells containing a genomic Tet-Bxb1-BFP landing pad by co-transfecting with Fugene 6 (Promega, Madison, WI) and Bxb1 recombinase expression vectors. The cells were then incubated at 33 C for 3 days and induced with Doxycycline (2 g/mL) one day after transfection. The cells were then incubated at 37 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 g/mL) until confluency when they were divided into 15 cm dishes for the three drug trial replicates. 7-12 days post-transfection, cells were washed with 1X phosphate buffered saline (AthenaES, Baltimore, MD) and harvested with 1X 0.25% Trypsin-EDTA (Gibco, Grand Island, NY). CFTR 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). 600,000 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 15-25 million cells per quartile fraction for the downstream genetic analysis.