Measuring DNA replication timing of maize root tips with Repli-seq method
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. The overall goal of this project is to define such linkages in maize by exploiting the genetic and genomic resources of this species. We used an updated Repli-seq protocol to profile replication timing in Zea mays B73 root tips and compare it to other methods for profiling replication timing. Overall design: 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 0-1 mm root segments from the primary and the first two emerging seminal roots 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 stages of the mitotic S phase. 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 S-phase stages using an AF-488 antibody (Invitrogen # A-11094, lot 2551344). 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.