Genome-scale mapping of replication origins by nascent strand (NS) chip. (A) NS isolation schematic. 0.5–2.5-kb NS were isolated from total genomic DNA by denaturation and sucrose gradient centrifugation. NS enriched by lambda exonuclease treatment were hybridized against total genomic DNA on high-density tiling arrays (see Supplementary Information). (B) Example of the distribution of replication origins in mouse (upper panel) and Drosophila cells (lower panel) along a 200-kb region. The log2-ratio between NS and total genomic DNA is shown. For genes, the position of the start site (high bar bordering the gene), exons (large gray boxes), and introns (thin gray boxes) are indicated (see Supplemental Fig. 2F). (C) Origin number and density per genome. (D) Immunoprecipitation of chromatin associated with ORC2 was carried out in P19 cells as described in Methods. Compilation of ORC2 signal strength data and correlation with the NS peaks is shown.
