Cell cycle progression and chromosome segregation assays
Structural basis of Stu2 recruitment to yeast kinetochores
eLife, Feb 16, 2021; DOI: 10.7554/eLife.65389

Cells were grown in YPD medium. Exponentially growing MATa cells with or without a tandem array of lacO sequences integrated proximal to CEN3 (Shonn et al., 2003) and a LacI-GFP fusion (Biggins et al., 1999; Straight et al., 1996) were arrested in G1 with 1 μg/mL α-factor. When arrest was complete, cells were released into medium lacking α-factor pheromone and containing 500 μM IAA and 0.05 μg/mL rapamycin at 23°C. For determining cell cycle progression,~75 min after G1 release, 1 μg/mL α-factor was added to prevent a second cell division and samples were taken every 15 min after G1 release to determine cell cycle state (via nuclear morphology of DAPI stained nuclei). To assess chromosome segregation in cells containing GFP labeled CEN3, we sampled cells during anaphase and determined the percent of missegretated chromosomes.