Replication timing by DNA copy number analysis, using DNA content flow sorting of G1 phase and S phase
During the cell cycle, DNA replication is a highly regulated 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 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.The terminal 0-1 mm root segments from B73 seedling roots were excised and separated into primary or seminal roots, 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 and total DNA was stained with DAPI. Nuclei were flow sorted based on DNA content only (DAPI fluorescence) for G1 and a broad S phase gate placed between the G1 and G2 DNA content peaks. Total genomic DNA was isolated from the G1 and S phase sorted nuclei, sheared and sequenced. The ratio of S phase 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.