title: GSM7884350: Library_induced_500k_cells_G1_gate; Saccharomyces cerevisiae; OTHER
library_construction_protocol: We sorted 1e6/500k cells into each pool. We extracted genomic DNA from these cells with the Zymo YeaSTAR (#D2002) kit. To ensure singular constructs per cell, we introduced our library into the URA3 locus of strain DHY211 (MATa, MKT1(30G,) RME1(INS-308A) TAO3(1493Q), CAT5(91M) MIP1(661T), SAL1+ HAP1+). Employing the established yeast transformation method (64), we subjected the transformation to 30 minutes at 30 °C followed by 60 minutes at 42 °C. To minimize potential PCR errors, we performed SalI and EcoRI digestion on the plasmid library, releasing the section encompassing the ACT1 promoter, the synthetic TF, and the KANMX marker. Simultaneously, PacI digestion was conducted to cleave plasmids devoid of an activation domain variant and barcode insert, thereby reducing the occurrence of transformants with inactive TFs. Directed integration into the URA3 locus was guided by 500 bp upstream homology spanning the URA3 and ACT1 promoters, along with a corresponding 500 bp downstream homology region spanning the TEF and URA3 terminators. These regions were PCR amplified from pMVS 295 (Strader 6161) and pMVS 296 (Strader 6768), a generous gift from Nick Moffy and Lucia Starder. Transformation utilized a molar ratio of 1:3 for linearized library to homology arms, with 28 μmol of linearized library per reaction. The transformed library was plated on YPD, followed by an overnight incubation at 30°C, and subsequent replica-plating onto freshly prepared SC G418 plates. Employing this process across 80 transformation reactions yielded an estimated >1,000,000 individual colonies. Subsequently, the transformants were collectively mated with an FY5 strain containing the reporter integrated into the uncertain ORF, YBR032w. Diploids were selected on YPD with G418 (200 µg/ml) and NAT (100 µg/ml) (strain MY436 YBR032w::P3_GFP NAT S288C), resulting in prototrophic diploids. These 110,000 yeast transformants were mated in batches, and prior to the final experiment, batches were pooled, and multiple aliquots were frozen. beta-estradiol inducible library