Unless otherwise stated, yeast cultures were grown using standard procedures (41 (link)) for 3 days at 30°C in the indicated media. Colonies on agar plates were pinned using a RoToR handling robot (Singer Instruments). For growth into stationary phase (starvation), synthetic complete medium (SC) with 0.1% glucose and an inoculation density of OD600 = 0.05 was used as starting condition. For exponential growth, cells were grown in SC with 2% glucose to an OD600 of 0.5–1.0 for at least 8 h.
For Thiolutin treatment the cells were grown in stationary phase for 24 h in liquid SC medium with 0.1% glucose at 30°C. Thiolutin (Abcam) was added to final concentration of 50 μM (from 10 mM stock in dimethyl sulfoxide) and aliquots of cells (10 ml) were taken with 12 min intervals for RNA extraction.
Yeast strain construction was performed using standard protocols for yeast transformation and polymerase chain reaction (PCR) targeting (42 (link),43 ) and strain validation using colony PCRs, in some cases followed by sequencing. Strains are listed inSupplementary Table S1 . Antisense library strains were published previously (40 (link)). Plasmids were constructed using standard procedures (44 ) and verified by sequencing. All plasmids are listed in Supplementary Table S2 .
For Thiolutin treatment the cells were grown in stationary phase for 24 h in liquid SC medium with 0.1% glucose at 30°C. Thiolutin (Abcam) was added to final concentration of 50 μM (from 10 mM stock in dimethyl sulfoxide) and aliquots of cells (10 ml) were taken with 12 min intervals for RNA extraction.
Yeast strain construction was performed using standard protocols for yeast transformation and polymerase chain reaction (PCR) targeting (42 (link),43 ) and strain validation using colony PCRs, in some cases followed by sequencing. Strains are listed in