GSM7893307: R1-MN-rep2; Mus musculus; MNase-Seq
1×107 cells were pelleted in PBS and then lysed in 1ml buffer A (10 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2; 1 mM DTT, 0.1% NP-40) on ice 10 min. Then the cells were centrifuged at 1,000g 5 min and resuspended in 1 ml buffer A without NP-40. The cells were incubated at 37°C 5 min. Digestion was initiated by adding 2 mM CaCl2 (final concentration) and 1 μl 200U/ml MNase and incubated at 37°C. 200 μl digested chromatin was taken out at the time point of 1, 2, 4 min and the reactions were stopped by adding 4μl 500 mM EGTA (final concentration 10 mM). the mixture was treated with RNaseA 1hr at 37℃, and proteinase K 2 hrs at 55℃. DNA was recovered by phenol-chloroform extraction and precipitated by ethanol, and separated by 1.2% agarose gel. The mononucleosome-sized DNA fragments were recovered by gel extraction kit (Qiagen) and the libraries were generated using the NEB Next ultra DNA library Prep kit for Illumina. The DNA libraries were purified with 1 volume AMpure beads and sequenced on Nova-seq6000 with paired-end 150 bp (Illumina).