Preparation of Fluorescently Labeled Nucleosomal DNA

Prior to polymerase chain reaction (PCR) amplifications of nucleosomal DNA, primers containing internal amino modified thymines (IDT, Supplementary Table S1) were labeled with Cy5-NHS ester (GE healthcare) and purified by reverse phase high-pressure liquid chromatography (RP-HPLC) on a C18 column (Agilent Technologies). Using a pair of Cy5-labled and unlabeled primers (Supplementary Table S1) with a Widom 601 nucleosome positioning sequence (601 NPS, Supplementary Table S2) containing plasmid (pDrive-601 NPS) as the template, the 601 NPS Fwd/Rev Entry-Exit–Cy5 and the 601 NPS Dyad–Cy5 DNA constructs were amplified by PCR (18 (link)). Similarly the 5S NPS Fwd Entry-Exit–Cy5 DNA construct was amplified by PCR from a plasmid (pBSII SK(-)-5S rDNA NPS) containing the Xenopus borealis 5S rDNA NPS sequence (Supplementary Table S2) (18 (link)). The reverse primers used in the PCRs were designed to incorporate biotin modified 78 base pair (bp) extensions to the 3′ end of each NPS (Supplementary Table S2 and Figure S1). After PCRs, the DNA constructs were purified by ion-exchange HPLC on a Gen-Pak Fax column (Waters).