Arabidopsis thaliana Col-0 cell culture replication timing. Arabidopsis thaliana

Arabidopsis thaliana Col-0 cell culture were pulse-labeled with the thymidine analog 5-Ethynyl-2'-deoxyuridine (EdU) for 10 minutes to allow actively replicating DNA to incorporate the EdU. The cell cultures were fixed in 1% formaldehyde for 10 minutes, washed in PBS, and snap frozen. Nuclei were isolated from the frozen cells, 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 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 three S-phase stages using an AF-488 antibody. The resulting IP DNA from three S-phase stages, and the G1 reference genomic DNA were Illumina sequenced to generate whole-genome DNA replication timing profiles.