Table 1.

Detailed outline of Synthetic Genetic Array (SGA) screen procedure

Description	Media	Agar or liquid media	Time
Prepare a lawn of query strain (VJY355)a	YPD	Agar	2 days
Pin array of library strains from 96-well plate of cryopreserved yeast	YPD	Agar	2 days
Mate yeast by replica-pinning query strain and library strains onto fresh plate	YPD	Agar	1 day
Mated diploid selectionb	YPD + 100 μg/ml nourseothricin (clonNAT; WERNER BioAgents) + 200 μg/ml G418 (G418 Sulfate; CALBIOCHEM)	Agar	2 days
Sporulationc	1% Potassium Acetate, 0.1% yeast extract, 0.05% glucose, 0.002% adenine, 0.004% uracil, 0.0005% arginine, 0.00025% histidine, 0.0015% isoleucine, 0.0015% leucine, 0.001% lysine, 0.00025% methionine, 0.0015% phenylalanine, 0.00125% threonine, 0.001% tryptophan	Agar	5–7 days
Haploid MATa selectiond	0.67% yeast extract without amino acids, 2% glucose, 0.0005% arginine, 0.00025% histidine, 0.0015% isoleucine, 0.0015% leucine, 0.001% lysine, 0.00025% methionine, 0.0015% phenylalanine, 0.00125% threonine, 0.001% tryptophan, 0.004% adenine, 0.008% uracil, 50 μg/ml canavanine; 50 μg/ml thialysine	Agar	2 days
Gene deletion or hypomorphic allele selectione	MATa selection media + 200 μg/ml G418	Agar	2 days
Selection of deletion or hypomorphic allele AND Deg1-Sec62-His3:natMX4 reporterf	MATa selection media + 200 μg/ml G418 + 100 μg/ml nourseothricin	Agar	2 days
Prepare 96-well plates with liquid cultures	MATa selection media + 200 μg/ml G418 + 100 μg/ml nourseothricin	Liquid	2 days
Transfer 40 μl of liquid culture to fresh media to select for genes required for Deg1-Sec62-His3 degradation	MATa selection media + 200 μg/ml G418 + 100 μg/ml nourseothricin without histidine	Liquid	11 h (Record OD595 at beginning and end of 11 h)

^aAll steps were performed at 30 °C, except for mating and sporulation, which were performed at 23 °C. All transfers (except final transfer to screen media) were performed using sterile 96-pronged pinners.

^bThe query strain locus encoding Deg1-Sec62-His3 also contains the natMX4 nourseothricin-resistance gene. All deletion and hypomorphic library strains possess the kanMX4 gene, which confers resistance to G418. Only mated diploid strains may grow in the presence of both nourseothricin and G418.

^cSporulation was induced by culturing yeast on media with limited nitrogen and carbon (106).

^dHaploid selection was mediated by toxic amino acid analogs thialysine and canavanine, which enter yeast viaLYP1 and CAN1 gene products, respectively. The query strain possesses LYP1 and CAN1 deletions, while screened library strains possess wild type alleles of these genes. Heterozygous LYP1/lyp1Δ CAN1/can1Δ mated diploid yeast are susceptible to thialysine and canavanine. Only haploid lyp1Δ can1Δ yeast can survive on such media. Furthermore, in the query strain, the CAN1 gene was replaced with the LEU2 gene driven by the promoter for the MATa-specific gene, STE2, which allowed only MATa cells to produce leucine and survive in the absence of exogenously provided leucine.

^eContinued presence of G418 ensures preservation and selection of yeast with library deletion or hypomorphic alleles.

^fContinued presence of nourseothricin ensures preservation and selection of yeast possessing Deg1-Sec62-His3:natMX4.