Genome-wide replication timing analysis identifies a subset of BAF250a sensitive replication domains. (A) Flow chart of genome-wide replication timing analysis . BrdU-substituted DNA from early and late S phase cells was differentially labeled and hybridized to a whole-genome oligonucleotide microarray. (B) Replication timing profiles from mock- and 4-hydroxytamoxifen (OHT)-treated BAF250a flox/flox embryonic stem cells (ESCs) for chromosome 15. The signal ratio of early and late (Log2 early/late) for each probe is plotted against the chromosomal position. Shown are loess-smoothed plots for the average of two biological replicate experiments. (C) Summary of significant early to late (EtoL) and late to early (LtoE) switching segments in OHT-treated cells. Replication timing data were averaged into 200-Kb windows and statistical significance was calculated between mock and OHT-treated ESCs, as described in Methods. (D) Expanded plots for exemplary regions that undergo replication-timing switches in response to BAF250a loss are shown below. (E) BAF250a-dependent timing switching domains align to developmental boundaries. Replication timing plots of exemplary EtoL and LtoE switching domains shown in Figure 1D (mock-red and OHT-blue) are compared to replication timing profiles from other cell types (gray). The top table shows the percentage of EtoL and LtoE switching domains that align to developmental boundaries. (F) Box plots show the sizes of EtoL and LtoE timing switching domains compared to non-switching domains. (G) Correlation of replication timing datasets (Pearson’s R values).