ORI chromatin segregates from bulk chromatin in density gradients. (A,B) Cross-linked chromatin was fractionated by CsCl density-gradient centrifugation into 16 fractions and analyzed by gel electrophoresis. The densities (g/cm³) of fractions 1–16 are 1.23, 1.24, 1.26, 1.28, 1.30, 1.32, 1.34, 1.37, 1.39, 1.42, 1.45, 1.49, 1.52, 1.57, 1.62, and 1.67, respectively. (A) Protein distribution (Coomassie staining of 8% SDS PAGE). (B) DNA distribution (ethidium bromide staining of 1% agarose). (C) Western blot showing the distribution of ORC1 and 2, MCM2, 5, and 7 in the gradient fractions. (D) Q-PCR distribution of DNA fragments from the LMNB2 and PRKDC ORIs and flanking regions (LMNB2: −1, +3 kb; PRKDC: +1, +5 kb), expressed as “fold enrichment” over input DNA of each fraction. (E) Q-ChIP of ORC1 binding at LMNB2 and PRKDC ORIs using anti-ORC1 or anti-Flag (Mock) antibodies from total chromatin or low-density fractions (6, 7). (F) ChIP-seq profile of ORC1 at LMNB2 and PRKDC ORIs. The position of the Q-PCR probes used in E is shown below each ORC1 peak. (Scale bars) 1 kb. Illumina sequencing was performed with purified DNA from anti-ORC1 ChIP of fractions 6 and 7 (red empty box) or with total DNA of fractions 14–16 (shown in Supplemental Figs. S1, S2).