MNase Digestion and Restriction Enzyme Accessibility

Micrococcal nuclease (MNase) digestion was performed as described (13 (link)). Nuclei isolated from ESCs were digested with 5 and 30 U of MNase (Worthington, Lakewood, NJ, USA, www.worthington-biochem.com) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight. The purified DNA was subjected to Southern blot analysis. Restriction enzyme accessibility assays were carried out as described (13 (link)). Isolated nuclei from ESCs were digested with 100 U of SapI, AlwI, FokI, XbaI, PstI, EcoRI or StuI (New England Biolabs, Ipswich, MA, USA, www.neb.com) for 30 min. The purified genomic DNA was re-digested with 100 U of AvaI (for Nanog gene) or HindIII (for Oct4 gene). The digested fragments were analyzed by Southern blot using ³²P-labeled probe (Figure 5A). All probes were listed in Supplementary Table S1.