TreatmentAgent

Auxin treatment (500 mM)

HCT116 RAD21-mAID cells were synchronized and treated as in (Oldach and Nieduszynski 2019 PMID: 30836708). 2.5 mM Thymidine was added to the media for 24 hours followed by wash out and a 3-hour release. 100 ng/mL nocodazole was then added to the media for 8 hours and mitotic cells were collected by shake off. Percentage of mitotic cells was estimated by metaphase spread. Collected cells were released into fresh media and 500mM 3-Indoleacetic acid (Auxin, I2886 Sigma) was added 30 minutes after release to degrade RAD21. Cells were released into the cell cycle for 4, 6, 8, and 10 hours. 400uM BrdU was added to the media 30 minutes before each time point after which the cells were collected and fixed in ethanol. Equal numbers of cells from each time point were pooled together for high resolution repli-seq sorting. High resolution repli-seq was performed as in (Zhao et al 2020 PMID: 32209126).