title: GNRHR DMS LIBRARY, W107A background, replicate 1, bin D
design: The expression of transiently expressed single mutants was carried out using a previously described pcDNA5 FRT expression vector containing mGnRHR cDNA with an N-terminal hemagglutinin (HA) epitope and bicistronic eGFP (Fig. 2) (6). Mutations were introduced by site-directed mutagenesis with PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga, Japan). Biochemical measurements of the membrane integration of TMD6 were carried out using a previously described pGEM vector containing modified leader peptidase (Lep), with TMDs of interest cloned into the H-segment (Fig. S1) (6, 13). Mutations were introduced into the H-segment by site-directed mutagenesis with PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga, Japan). Plasmids were purified using a ZymoPURE Midiprep Kit (Zymo Research, Irvine, CA). DMS experiments were carried out using a previously described pcDNA5 FRT vector containing mGnRHR cDNA bearing an N-terminal influenza hemagglutinin (HA) epitope, followed by an internal ribosome entry site (IRES) and eGFP sequence, which was further modified to be compatible with recombination-based DMS approaches (6). First, an attB recombination site was inserted in place of the CMV promoter by InFusion HD Cloning (Takara Bio, Shiga, Japan). To facilitate the creation of these libraries using nicking mutagenesis, a BbvCI restriction site was introduced by site-directed mutagenesis using PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga, Japan). The V276T and W107A mutations were also introduced by site-directed mutagenesis with PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga, Japan). To generate mutational libraries, a 10N unique molecular identifier (UMI), or barcode, was first inserted into the plasmid backbone using a previously described nicking mutagenesis method (46). Nicking mutagenesis plasmid products were transformed into NEB 10-beta electrocompetent cells (New England Biolabs, Ipswich, MA) and purified using a ZymoPURE Midiprep Kit (Zymo Research, Irvine, CA). A programmed oligo pool (Agilent, Santa Clara, CA) encoding all 19 amino acid substitutions at 85 residues throughout the mGnRHR sequence was then used to generate pools of single and double mutants in the context of WT, V276T, and W107A mGnRHR cDNA using a previously described nicking mutagenesis method (21). The resulting plasmid products were transformed into NEB 10-beta electrocompetent cells (New England Biolabs, Ipswich, MA) and purified using a ZymoPURE Midiprep Kit (Zymo Research, Irvine, CA). To limit the number of mGnRHR variants per UMI, these libraries were then bottlenecked through an additional transformation in NEB Turbo electrocompetent cells (New England Biolabs, Ipswich, MA), and were again purified using a ZymoPURE Midiprep Kit (Zymo Research, Irvine, CA). The resulting plasmid preparations contained detectable levels of a concatemer product, which were removed from the mutational libraries by gel purification using a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Purified products were again transformed into either NEB 10-beta (New England Biolabs, Ipswich, MA) or SIG10 (Sigma-Aldrich, St. Louis, MO) electrocompetent cells, and purified with a ZymoPURE Midiprep Kit (Zymo Research, Irvine, CA). To associate each UMI with its corresponding GnRHR variant, the final plasmid libraries were also sequenced using PacBio SMRT sequencing. Briefly, plasmid libraries were first double-digested with PmlI and MfeI-HF restriction enzymes (New England Biolabs, Ipswich, MA), then purified using a Zymo DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA). Complete digestion was verified by analyzing the products on an agarose gel, purity was assessed with a Synergy Neo2 microplate reader (BioTek, Winooski, VT), and the final yield was quantified using a Qubit4 fluorometer (ThermoFisher, Waltham, MA). The 1537 bp fragment for each library, containing the 10N UMI and GnRHR open reading frame, was isolated on a BluePippin instrument (Sage Science, Beverly, MA). These fragments were then assembled into PacBio libraries and sequenced on the Sequel II system with a 30-hour runtime. The sequence of each plasmid-derived fragment containing mGnRHR variants and their corresponding UMIs was determined by reconstructing the circular consensus sequences (CCS) for each independent well in the PacBio readout. mGnRHR libraries were sequenced to a depth of 1,825,010 wells for WT, 1,545,151 wells for V276T, and 1,497,359 wells for W107A. A minimum coverage of five passes around each circular fragment (subread) was required for incorporation of sequencing data from an individual well into the analysis. CCS were mapped to the reference open reading frame using minimap2. CCS that did not contain a complete 10-base UMI sequence or fully cover the target ORF were excluded from the analysis. Variations in the mGnRHR sequence of individual fragments were called using Samtools. Reads were then grouped according to their UMI sequences. Given that insertion and deletion (indel) artifacts are prevalent within PacBio sequencing data, we only removed UMIs from the analysis if indels were detected at a specific position within the UMI in at least 10% of the subreads. Indels observed within the open reading frame were only called if their occurrence was judged to be statistically significant relative to the background indel rate for the reads according to a binomial test (p <= .05). Codon substitutions encoded in our primer library (Table S3) were called within the mGnRHR open reading frame only if they were detected in at least 75% of the individual subreads. Only UMIs that were found to be associated with a single mGnRHR variant were included in the final analysis. Our final dictionaries for the WT, V276T, and W107A libraries contained 3,261, 531, and 4,962 unique UMIs, respectively, that could be indexed to a corresponding mGnRHR variant. A previously described HEK293T cell line with a genomic Tet-Bxb1-BFP landing pad was obtained from D. Fowler (18). Cells were grown at 37 oC and 5.0% CO2 in complete media, made of Dulbeccos Modified Eagles medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY), 0.5% penicillin (Gibco, Grand Island, NY), and 0.5% streptomycin (Gibco, Grand Island, NY). 2 million cells were plated in 10-cm tissue culture plates and co-transfected the next day with 475 ng of plasmid encoding bxb1 recombinase and 7125 ng of the plasmid library using Lipofectamine 3000 (Invitrogen, Carlsbad, CA).  Cells were grown at 33 oC for 4 days after transfection. 2 days after transfection, the cells were induced with 2 g/mL doxycycline. All cells were expanded into 15-cm tissue culture plates 6 days after transfection, and 10 million cells were re-plated into new 15-cm tissue culture plates 3 days later. Twelve days after transfection, the cells were washed with 1X phosphate-buffered saline (PBS, Gibco, Grand Island, NY), and harvested with TrypLE Express protease (Gibco, Grand Island, NY).  Cells were washed twice with 2% fetal bovine serum in PBS (wash buffer), resuspended in PBS containing 2% FBS to 10M cells/ mL, and then sorted on a BD FACS Aria II (BD Biosciences, Franklin Lakes, NJ). Recombined cells were isolated based on their characteristic gain of bicistronic eGFP and loss of BFP expression (18). Forward and side scatter profiles were first used to gate for intact live cells, and cells with eGFP-positive (488 nm laser, 530/30 nm emission filter) and BFP-negative (405 nm laser, 450/50 nm emission filter) fluorescence profiles were collected in complete media supplemented with 10% FBS (Gibco, Grand Island, NY) and then plated in 10-cm tissue culture plates. Cells were induced with 2 g/mL doxycycline 24 hours after sorting. 4 days after sorting, 10 million cells were passaged into 15-cm tissue culture plates. 7 days after sorting for eGFP-positive cells, cells were sorted again according to the surface expression of GnRHR variants. Briefly, cells were first washed with 1X phosphate-buffered saline (PBS, Gibco, Grand Island, NY), and harvested with TrypLE Express protease (Gibco, Grand Island, NY). GnRHRs expressed at the cell surface were immunostained for 30 minutes in the dark with DyLight 550-conjugated anti-HA antibody (Invitrogen, Carlsbad, CA). Cells were then washed twice with PBS containing 2% FBS, and sorted on a BD FACS Aria II (BD Biosciences, Franklin Lakes, NJ). Forward and side scatter profiles were used to gate for intact live cells, and eGFP fluorescence was used to gate for recombined cells. Cells were then sorted into even quartiles based on the amount of DyLight 550 fluorescence (561 nm laser, 585/15 nm emission filter). Sorted cells were collected in complete media supplemented with 10% FBS (Gibco, Grand Island, NY) and plated in 10-cm tissue culture plates. Cells were allowed to grow to confluency and then were washed with 1X phosphate-buffered saline (PBS, Gibco, Grand Island, NY) and harvested with TrypLE Express protease (Gibco, Grand Island, NY). Cell pellets were frozen for subsequent analysis. Genomic DNA (gDNA) was extracted from cell pellets using the Sigma GenElute Mammalian gDNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO). DNA amplicons for Illumina sequencing were produced from the gDNA using a previously described semi-nested polymerase chain reaction (PCR) technique (18). In order to selectively amplify recombined DNA, the first PCR utilized a primer which anneals upstream of the 10N UMI and a primer which anneals in the BFP-encoding region of the landing pad. Eight replicate PCRs were carried out with 2.5 g of gDNA and HiFi HotStart Ready Mix (Kapa Biosystems, Wilmington, MA). These PCRs were limited to 7 cycles to avoid PCR bias. PCR products were purified using a ZR-96 DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA), and replicate products were then pooled. 10 L of this first PCR product was then used as the template for a second PCR, which utilized primers that incorporate Illumina adapter sequences to both ends of the 10N UMI. To avoid PCR bias, these reactions were monitored by real-time PCR on a StepOne RT-PCR instrument (Applied Biosystems, Waltham, MA) and terminated during mid-log amplification (17 or 18 cycles). Four replicate PCRs were carried out with HiFi HotStart Ready Mix (Kapa Biosystems, Wilmington, MA) and SYBR Green fluorescent dye (Applied Biosystems, Waltham, MA). Replicate PCR products were then combined and purified with a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). To further improve the yield and quality of the amplicons, an additional 6-cycle PCR was carried out with the HiFi HotStart Ready Mix (Kapa Biosystems, Wilmington, MA) and primers that preserve the DNA sequence. These PCR products were then purified using a Zymo DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA). Finally, amplicon yield and quality were assessed using an Agilent 2200 TapeStation with D1000 tape (Agilent Technologies, Santa Clara, CA) and a Synergy Neo2 microplate reader (BioTek, Winooski, VT). Amplicons were sequenced using an Illumina NextSeq150 flow cell, paired end, with 50% PhiX content. This particular library consists of the paired end sequencing reads (R1 and R2) obtained from replicate 1 from the W107A background, quartile 4 (bin D).