Fig. 2: Simplified representation of experimental workflow.

Example of a single cross plate where two MATa ‘bait’ strains in which the gene of interest (GOI) was knocked out (KO) with a kanamycin resistant marker (kanMX, also confers resistance to G418) are each crossed to the 36 MATa ‘hit’ strains in which the gene of interest was knocked out with a nourseothricin-resistant marker (natMX). Heterozygous diploids were selected for on media containing both antibiotics, and then sporulated on standard sporulation media. The sporulated colonies were pinned onto a series of specialized SGA media that select for MATa and MATα haploid progeny. Positions on the double mutant haploid plates that would have resulted in ‘monogenic crosses’ (where the same gene of interest was knocked out in both parents) or ‘single parent crosses’ (where one of the parent positions was empty) were monitored for potential false-negatives. The double mutant haploid progeny were used to identify synthetic lethal interactions and then pinned in quadruplicate on a fresh YPD plate. WT controls were added, and the resulting master plate was pinned onto six different media types for phenotyping. Phenotyping plates were imaged every 12 h to monitor growth rates.