Yeast strains were cultured overnight in 4 ml YPD at 30 °C. Cells were subcultured for 4–5 h and isolated during exponential growth. Overall, 400 μl of cells were pelleted and fixed overnight in 70% ethanol at 4 °C. The fixed cells were washed twice with 1 ml of sodium citrate (50 mM) and resuspended in 500 μl sodium citrate and 12 μl of RNaseA (25 mg/ml). The cells were incubated at 37 °C for 4 h. Then 17 μl of propidium iodide was added to the RNase-treated cells and incubated overnight at 37°C. The cells were vortexed and sonicated for 5 s at level 15 of the Sonic Desmembrator (Fisher Scientific), and filtered immediately before analysis using a BD Falcon tube with a 35 μm nylon mesh cap (Cat. No. 352235). Ploidy was determined using a Yetti flow cytometer, and 50,000–100,000 cells were analyzed per strain.