Pcr purification kit

Mouse 3134 cells were cultured, treated with vehicle or 100 nM dexamethasone for 1 h prior to harvest, and nuclei were prepared as described (23 (link)), except for the following modifications. Prior to MNase digestion, nuclei were washed in Nuclei Suspension Buffer (25% glycerol, 5 mM Mg-acetate, 5 mM HEPES pH 7.3, 0.08 mM EDTA, 0.5 mM spermidine, 1 mM DTT) and resuspended in MNase Digestion Buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl₂, 1 mM CaCl₂, 0.5 mM spermidine) at 75 million nuclei/ml and 400 U/ml MNase (Worthington). Nuclei were digested in 400 ul aliquots for 4–7 min at 37°C with gentle agitation; the reaction was terminated with Stop Buffer (500 mM NaCl (140 mM final), 50 mM EDTA (14 mM final), 20 mM EGTA (5.6 mM final), 3.6% SDS (1% final)) to yield ∼90% mono-nucleosomes (Supplementary Figure S1). The digests were treated with RNase (Life Technologies) for 30 min, then proteinase K (Ambion) for another 30 min. Samples were extracted twice with phenol/chloroform and ethanol-precipitated. DNA pellets were washed twice with 70% ethanol, dried and dissolved in water. Nicks in MNase-digested DNA were repaired using the PreCR repair mix (NEB) and the DNA was purified using a PCR purification kit (Life Technologies).