Genome-wide binding of the meiosis specific Rec8 cohesin subunit in metaphase I

ChIP-seq profile of Rec8 binding sites arrested in metaphase I by depletion of the APC/C activator CDC20. The aim of this experiment is to identify Rec8 binding sites in metaphase I. Diploid S. cerevisiae SK1 cells were constructed containing Rec8-3Ha in an pGAL1-NDT80 pGPD1-GAL4.ER pCLB2-CDC20 background. Cells were allowed to sporulate in sporulation media for 5 hours before addition of β-estradiol to release from prophase arrest. Cells were grown for 75min to arrest in metaphase I after which formaldehyde was added to the cultures and cells were fixed for 30min. We chose this double-arrest strategy (with a prophase I arrest followed by a metaphase I arrest) to ensure efficient arrest in metaphase I, which is less robust in cells without Spo13. Samples were subsequently processed according to the standard lab ChIP-Seq protocol.