Generation of replication timing profiles. (A) Repli-Seq cell sorting. BrdU pulse-labeled cells were sorted into four S-phase fractions by flow cytometry: early, early-mid, mid-late, and late. BrdU-labeled DNA was isolated from the flow-sorted cells and active replication intermediates were precipitated with anti-BrdU antibodies and sequenced by next-generation sequencing. (B) Distribution of BrdU-labeled sequences in Kc167 cells. The read depth (RPKM) from each fraction is shown for a 4-Mb portion of chromosome 2L. The early (E, red) and late (L, blue) fractions are enriched in largely nonoverlapping locations with the middle fractions (early-mid [E–M], green; late-mid [L–M], purple) displaying an intermediate pattern changing from E to L. (C) Replication timing profiles for Kc167 (black), S2 (gray), and DmBg3 (orange) cells. Replication timing ratios were derived from the four fractions to generate a relative timing value for each genomic position. High values represent early replicating regions and low values represent late replicating regions. A representative 4-Mb region on chromosome 2L is shown. (D) Replication profiles are conserved but not identical between cell lines. Pairwise Pearson correlation for the replication timing values was determined for each pair of cell lines.
