Deep mutational sequencing of hOCT1 (SLC22A1): VAMPseq with GFP-tagged OCT1, FACS bin 3, replicate 3
The OCT1 library was generated using an oligo pool-based approach, where designed variants are encoded in oligos and then full-length libraries are assembled with Golden Gate sites. The final library were assembled in a landing pad cell line compatible backbone containing a Carbenicillin resistance cassette and GSGSGS-mNeonGreen Fragment P2A-Puromycin cassette for positive selection. Libraries were cloned into a landing pad vector containing a BxB1-compatible attB recombination site using BsmBI mediated golden gate cloning. We designed the landing pad vector which we recombined the library into to contain BsmBI cutsites with compatible overhangs for the library to have an N terminal kozak sequence and in-frame with a C-terminal GSGSGS-mNeonGreen Fragment P2A-Puromycin cassette for positive selection. To make the cell lines, 1500 ng of library landing pad constructs were co-transfected with 1500ng of a BxB1 expression construct (pCAG-NLS-BxB1) using 10.5L of lipofectamine LTX in 10 wells of a 6 well plate. For VAMPseq, samples were taken and genomic DNA was extracted using a Quick-DNATM Microprep Plus Kit from cells sorted into four different GFP intensities. The extracted genomic DNA was amplified using primers specific for hOCT1. Amplicons were then prepared for sequencing using the Nextera XT DNA Library kit from Illumina with 1 ng of DNA input. Samples were indexed using the IDT for Illumina Nextera DNA Unique Dual Indexes and SPRISelect beads at a 0.9x ratio were used for cleanup and final size selection. Each indexed tagmented library was quantified with Qubit HS as well as Agilent 2200 TapeStation. Samples were then pooled and sequenced on a NovaSeq 6000 SP300 flowcell in paired-end mode, generating fastq files for each sample after demultiplexing.