Zea mays

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 0-1 mm or 1-3 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 early, mid and late stages of either the mitotic S phase (0-1 mm) or the endocycle S phase (1-3 mm). G1 nuclei were also sorted to use as a genomic DNA reference for sequencing. After DNA was isolated from the sorted nuclei, EdU/AF-488 labeled DNA was immunoprecipitated from each of the three S-phase stages using an AF-488 antibody. The resulting IP DNA from the S-phase stages, and the G1 reference genomic DNA were Illumina sequenced to generate whole-genome DNA replication timing profiles.