Dissection and Genotyping of Yeast Meiotic Tetrads

For analysis of the meiotic product, we crossed a MATa with a MATalpha haploid strain, selected for diploids based on auxotrophy or antibiotic resistance, and patched the diploid strain on rich pre-Sporulation plates (YP agar with 6% [w/v] glucose]. Then we froze part of the patch and transferred part of the patch into Sporulation medium (1% potassium acetate, 0.005% zinc acetate buffer) and incubated the cultures with shaking at 23 °C. After a few days, the sporulation cultures were treated in a ratio of 1:1 [v/v] with Lyticase (L4020 Sigma Aldrich, 2.5 mg/ml, 200 units/µl, in 1 M D-Sorbitol) to digest the ascus. After 15–20 min at room temperature, the culture was applied to an agar plate and tetrads were dissected using a Singer micromanipulator. Colonies of haploid spores grew at 30 °C for three days. Images were taken at 48 and 72 h with the ChemiDoc™ Touch Imaging System (Bio-Rad). After three days, the spores were replica plated, genotypes were scored and strains were frozen in 15% glycerol-containing cryopreserved stocks at −80 °C. Strains are listed in Supplementary Data 2.