To deliver each PE2 variant to cell library A or B, PE2 variant-, pcDNA-BSD-, and puro-eGFP-encoding plasmids were mixed at a weight ratio of 10:1:1 to yield a total of 12 μg (for experiments using library 1) or 24 μg (for library B) of plasmid mixture, which was then transfected into a total of 1 × 106 cells from cell library A or a total of 6 × 106 cells from cell library B using Lipofectamine 2000 (Invitrogen), following the manufacturer’s protocol. After incubation overnight, the culture medium was exchanged with DMEM containing 10% FBS and 40 μg/ml blasticidin S (InvivoGen). Five days later, the transfected cells were harvested with 0.25% trypsin for genomic DNA extraction and deep sequencing.
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