Deep mutational sequencing of WT Met kinase: IL3 withdrawal, timepoint 1, replicate 2
TPR-MET-IRES-mCherry and TPR-METEx14-IRES-eGFP were retrovirally transduced and expressed in Ba/F3 cells for a proliferation screen in the absence and presence of interleukin-3 (IL-3). Genomic DNA (gDNA) was isolated from cells using the TakaraBio NucleoSpin Blood QuickPure kit. The target library was PCR amplified from 5g gDNA for each sample using the TakaraBio PrimeStar GXL system, with a final forward and reverse primer concentration of 0.3uM using the following parameters: initial denaturation at 98C for 30s, 24x cycles of denaturation at 98C for 10 sec, annealing at 60C for 15 sec, extension at 68C for 14 sec, and a final extension at 68C for 1 min. The purified libraries were gel purified (0.8% agarose, 1X TBE) with the Zymo Gel DNA Recovery kit and quantified by a Qubit dsDNA HS assay. Amplicons were  prepared for deep sequencing with the Nextera XT DNA Library Prep and indexed using IDT for Nextera Unique Dual Indexes (Set C). Indexed samples were quantified with TapeStation HS D5000 reagents, normalized, pooled, and paired-end sequenced (SP300) on two lanes of a NovaSeq6000 flowcell.