Transposon mutagenesis libraries for forward genetic screens. A) Diagram of PB plasmid pPB-SB-CMV-puro-SD. Inverted repeats (IRs) for the PB and SB transposons are shown. The cytomegalovirus enhancer and promoter is drawn as CMV. The rabbit β-globin splice donor is depicted with an arrow indicating its reading outward into adjacent genes. The construct is in a pBluescript-based plasmid vector. B) Transposase is required for transposon integration. Cells were transfected with PB plasmid in presence (+PBase), or absence (−PBase) of transposase plasmid followed by puromycin treatment. C) Transposition efficiency. Shown are PB transposition efficiencies with and without transposase. D) Splinkerette PCR template for insertion site detection. Nested PCR primers contain Illumina adaptors shown as red and green. A 6nt region in the linker (DDDDDD) serves as multiplexing barcodes. E) Mutagenesis and screen flow chart. The mutagenesis prescreened library was generated by transfection and expanded. Following drug selection, resistant samples were either isolated or pooled, and the insertion sites were identified by splinkerette PCR, Illumina sequencing, and mapping to a model genome.