Yeast Genetic Manipulation Protocols

All strains used in this study were derived from the haploid auxotrophic S288c strain BY4741 (MATa). A full list of strains used can be found in Table S1. Primers used for strain construction and verification were ordered from IDT. Primer sequences are listed in Table S2.
Deletion strains were constructed with a HygB cassette amplified from the pCB1 plasmid using primers with at least 40 bp sequence homology to the target DNA [74 (link)]. For fluorescent tagging, yECitrine was amplified from the pKT140 plasmid using primers with 40 bp sequence homology to the target DNA [75 (link)]. A list of plasmids used in this study and their genotype can be found in Table S3. PCR products were used for yeast transformation using a LiAc-based procedure. Transformants were verified by PCR using specific check primers.
Yeast was cultured in peptone dextrose (YPD) medium containing 2% bacteriological peptone (Lab M, Heywood, UK), 1% yeast extract (Lab M, Heywood, UK) and 2% glucose (Sigma, St. Louis, MO, USA). For solid plates 2% agar (Lab M, Heywood, UK) was added. YPD containing 200 µg/L Hygromycin B (Invitrogen, Waltham, MA, USA) or 200 µg/mL G418 (Formedium, Swaffham, UK) was used for selection of yeast transformants.