Nucleosome Reconstitution from 601 Sequence

The “601” nucleosome-positioning DNA sequence was used for nucleosomal template preparation (Lowary & Widom, 1998 (link)). The DNA template was amplified by PCR and fluorescently labeled using a Cy-5 conjugated reverse primer. The PCR product was purified with a PCR purification kit (QIAGEN) before nucleosome reconstitution. For nucleosome assembly, the DNA template was mixed with recombinant histone octamer in a 1:3 M ratio and a twofold DNA mass of sheared salmon sperm DNA (Ambion) and the mixture was then dialyzed in dialysis buffers (10 mM Tris–HCl, pH 7.5, 0.1% NP-40, 0.2 mM EDTA, and 5 mM 2-mercaptoethanol) containing various salt concentrations (2, 1.5, 1, 0.75, 0.5 M, and 10 mM NaCl, respectively) from high to low salt stepwise as described previously (Hsieh et al, 2015 (link)).