Synthetic Genetic Array Protocol

Three independent biological replicates of each SGA were performed with the Bioneer V5 deletion collection^{40 (link)} using a ROTOR HDA pinning robot (Singer Instruments) as previously described^{41 (link)}. Briefly, the three query strains h-btn1::NatMX ura-D18 leu1-32 ade6-M210, h-btn1^102–208del::NatMX ura-D18 leu1-32 ade6-M210, h-btn1^D363G::NatMX ura-D18 leu1-32 ade6-M210 and a control query strain, h-ade6::NatMX ura-D18 leu1-32 ade6-M210^{42 (link)} were mated with the library on Edinburgh Minimal Medium without nitrogen (Formedium). Following sporulation for 3 days at 25 °C and spore selection for 3 days at 42 °C, spores were pinned onto YES agar for 2 days at 32 °C to recover. Double mutant haploids were selected by growing cells on YES agar with ClonNat (Jena Bioscience; AB-102XL; 100 μg/ml) and G418 (Formedium; 500 μg/ml) for 2 days at 32 °C. Double mutant libraries were then grown on YES agar in quadruplicate (1536-well format) for 2 days at 32 °C.