8e7abfda-5536-11e8-a7b8-000e1e0af2dc
Zea mays B73, seedling root tip (0-3mm), sorted nuclei, CENH3 ChIP
B73 seedling roots were pulse-labeled with the thymidine analog 5-Ethynyl-2''-deoxyuridine (EdU) for 20 minutes to allow actively replicating DNA to incorporate the EdU. The terminal 0-3 mm root segments were excised, fixed in 1% formaldehyde for 15 minutes, washed in PBS, and snap frozen. Nuclei were isolated from the frozen, excised roots, the incorporated EdU was "clicked" to Alexa Fluor-488 (AF-488), and total DNA was stained with DAPI. Nuclei were flow sorted based on EdU incorporation (AF-488 fluorescence) and DNA content (DAPI fluorescence) into various cell cycle populations. Chromatin extracted from sorted nuclei was used for ChIP and the resulting DNA Illumina sequenced.