Cells were grown to a density of 3E6 cells/ml, and 1E8 cells from asynchronous cultures were used for each experiment.

Reads were mapped to the genome assembly using MAQ, allowing for up to 3 mismatches (quality score of at least 35). PeakSeq was used to make peak calls with a maximum q-val of 0.05 for each replicate. Replicates were combined by intersection: overlapping peak calls were considered verified and were reduced in a per-nucleotide union. The union of Orc2 binding sites from all 3 cell lines was taken to generate a set of meta-peaks; each meta-peak represents a contiguous region covered by an early activating origin of replication present in at least one cell line. In total, 7246 meta-peaks were called, 2395 common to all three cell lines.