DNA replication timing of Arabidopsis thaliana chromosome 4

DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about the replication proteins of plants. We used flow cytometry and isothermal Nimblegen arrays to profile DNA replication of Arabidopsis chromsome 4 during early, mid and late S phase. Replication timing profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes essentially all of the heterochromatin. Replicating DNA of cultured Arabidopsis Col-0 cells was labeled with BrdU for 1 h. After fixation using 1% formaldehyde, nuclei were isolated and flow sorted into Early, Mid and Late S phase based on DNA content (DAPI). Nascent DNA isolated by immunoprecitation to BrdU, amplified and labeled with Cy5/Cy3. Input DNA labeled with Cy5/Cy3 was the reference. Equal amounts of DNA was hybridized to an isothermal Nimblegen array that covered Arabidopsis chromosome 4. Each 2 phase sample has 2 biological replicates with a dye swap serving as a technical replicated for a total of 12 arrays.