Replication timing by DNA copy number analysis, using G1 phase and EdU-S 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 are using 5-ethynyl-2'-deoxyuridine (EdU) labeling and fluorescence activated nuclei sorting 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. The terminal 0-1 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.