Measuring DNA replication timing of maize root tips using DNA copy number (S/G1) method with an EdU lableing step for bivariate sorting of S and G1 phase

DNA replication is a highly regulated cell cycle process that integrates the time that a genome segment replicates in S phase with its transcriptional activity, chromatin structure, and epigenetic state. We use both DAPI and 5-ethynyl-2'-deoxyuridine (EdU) labeling to perform fluorescence activated nuclei sorting (FANS) in combination with high throughput sequencing to characterize the patterns of DNA replication across the maize B73 genome using a DNA copy number based procedure. 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. Then seedlings were rinsed and placed in 100 uM Thymidine to stop the label incorporation. The terminal 1 mm root segments from primary and seminal roots were excised, fixed in 1% formaldehyde for 15 minutes, quenched with glycine, washed in 1x 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) from a broad EdU-labeled S phase gate. Unlabeled G1 nuclei were also sorted. Total genomic DNA was isolated from the G1 and EdU-S phase sorted nuclei, sheared and sequenced. The ratio of EdU-S to G1 genomic DNA provides a measure of the population average DNA copy number for each genomic loci, where earlier replicating genomic regions will have a greater copy number than later replicating genomic regions.