Screen for GCR-suppressing genes and interacting genes

Query strains grown on YPD-agar were crossed to arrayed strains containing bait mutations on YPD-agar in quadruplicate by pinning onto a fresh YPD agar plate using a Singer RoToR robot (Singer Instruments, UK) and grown for 1–2 days at 30 °C. The cells were then subjected to two rounds of pinning onto diploid selection medium (YPD-agar containing 200 μg ml⁻¹ geneticin (G418; Gibco) and 100 μg ml⁻¹ nourseothricin (clonNAT; Werner BioAgents)) and grown for 1–2 days at 30 °C. The cells were then pinned onto presporulation medium (containing 15 g Difco nutrient broth (Fisher Scientific), 5 g Bacto-yeast extract (Fisher Scientific), 10 g Bacto-agar (Fisher Scientific) and 62.5 ml 40% glucose per 500 ml) and grown for 3 days at 30 °C. Cells from the presporulation medium were then pinned onto sporulation medium (10 g potassium acetate, 0.05 g zinc acetate, 20 g Bacto-agar per liter, containing a final concentration of 50 μg ml⁻¹ G418 and 25 μg ml⁻¹ nourseothricin) and incubated for 7 days at 30 °C. The resulting spore-containing cells were then subjected to two rounds of pinning onto diploid killing medium (1.7 g yeast nitrogen base without amino acids and without ammonium sulfate (Fisher Scientific), 1 g L-glutamic acid monosodium salt (Sigma), 2 g dropout mix³⁴ without uracil, lysine, leucine and, when appropriate, without histidine, 20 g Bacto-agar, 50 ml of 40% glucose per liter, containing a final concentration of 50 μg ml⁻¹ thialysine (S-(2-aminoethyl)-L-cysteine hydrochloride; Sigma), 10 μg ml⁻¹ cycloheximide, 200 μg ml⁻¹ G418, and 100 μg ml⁻¹ nourseothricin) followed by growth for 5 days at 30 °C for the first pinning and 2 days at 30 °C for the second pinning. Cells were then subjected to two rounds of pinning and growth on haploid selection medium (1.7 g yeast nitrogen base without amino acids and without ammonium sulfate, 1 g L-glutamic acid monosodium salt, 2 g CSM dropout mix without leucine, uracil and, when appropriate, without histidine, 20 g Bacto-agar, 50 ml of 40% glucose per liter, containing a final concentration of 200 μg ml⁻¹ G418 and 100 μg/ml nourseothricin) and grown for 2 days at 30 °C. Then the cells were pinned and grown on YPD-agar followed by storage at −85 °C in YPD media containing glycerol.