500μL samples were taken from yeast cultures (∼OD600 of 0.6). The cells were centrifuged at 3.000g for 2 min at RT. The supernatant was discarded and 1mL 70% Ethanol was added to the pellet. After thorough vortexing, the fixed cell suspensions can be stored at 4°C until further use. 500μL of the suspensions were transferred to a fresh tube and then centrifuged (3.000g, 2min RT). The supernatant was discarded and the pellet dissolved in 300μL 50mM sodium citrate and 0.1 mg/mL RNAse A. After 2h incubation at 50°C, proteinase K was added to a final concentration of 0.1 mg/mL, followed by another 2h incubation at 50°C. 30μL of this sample was then mixed with 170μL 50mM sodium citrate and 0.5μM Sytox Green (S7020, Thermo Fisher Scientific). Prior to FACS analysis, the samples were briefly sonicated (Bioruptor, 5 × 15sec) to detach cell clumps before proceeding with the analysis.
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