Figure 1. Generating and screening haploid transposon mutant libraries.
A. The piggyBac transposon used for mutagenesis. The transposon cargo contains splice acceptors that disrupt transcription, but gene trapping is not directly selected for. PuroΔTK, is a positive-negative selection marker: puromycin can be used to select for integrations, and FIAU to select for loss of the transposon [30]. B. Outline of the mutagenesis and screening process. A detailed protocol is provided in Protocol S1. C. The mutant pool remains predominantly haploid, as shown by propidium iodide staining of fixed cells from library H3L1. D. Determining drug concentration for screening. Olaparib was used at a concentration that kills >2.5×105 wild type haploid cells (4 µM). E. Scheme for further analysis of clones of interest by transposon reversion.