title: GSM1941477: Ep400 knockdown MNase-seq replicate 1; Mus musculus; MNase-Seq
library_construction_protocol: 1 million cells were fixed 10 min in culture medium containing 1% formaldehyde and ollected in 15 mM Tris-HCl PH7.5, 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5mM MgCl2, 0.1 mM EGTA, 0.4% Igepal CA-630 (Sigma). We next added 2 mM CaCl2 and 4 units of MNase, and incubated 10 min at 37°C. MNase digestion was stopped by adding 10 mM EDTA (final concentration), and storing on ice. Cells were then disrupted by 15 passages through a 25G needle, followed by a 10 min centrifugation at 18,000 g. The supernatant was collected and incubated 1h at 65°C with 15 mg of RNase A. We next added 10 mg of proteinase K, adjusted each sample to 0.1% SDS (final concentration) and incubated 2 h at 55°C. NaCl concentration was then adjusted to 200 mM and the samples were incubated overnight at 65°C for crosslink reversal. DNA was purified from each sample by phenol-chloroform extraction followed by ethanol precipitation. 20 ng of purified DNA was used for library preparation according to manufacturer's instructions, using Ultralow ovation library system, Nugen. Following end-repair and adapter ligation, fragments were size-selected onto an agarose gel in order to purify genomic DNA fragments between ~ 60 and 220 bp.
identifiers.namespace: GEO
identifiers.id: GSM1941477