Double-barcoded double-deletion yeast strain generation

Haploid gene deletion strains, carrying either KanMX or NatMX marked deletions, were derived from the diploid heterozygous deletion collection (Tong et al. 2001; Pan et al. 2004) for the following genes and dubious ORFs: ARP6, SAP30, SDS3, PHO23, SIN3, DGK1, SNT1, DEP1, RPD3, YHR095W, and YFR054C. Each of the 11 deletion strains marked with KanMX was mated to two unique strains from the 5′-barcode-construct-carrying yeast library. NatMX marked deletion strains were each mated to two strains from the 3′-barcode-construct-carrying yeast library. Resulting diploid strains from each cross, and carrying a deletion and the barcode construct, were sporulated and plated for haploid single colonies.

To obtain strains carrying two gene deletions and both complementary barcode constructs, all pairwise combinations of singly barcoded deletion strain were mated. In each resulting diploid, Cre-mediated recombination was induced at the barcode locus by growing on SC + 2% Galactose –Ura at 30° for 2 d. Cells were sporulated, and unsporulated diploids were digested using zymolyase as described (Herman and Rine 1997) before selecting single haploid colonies.