title: GSM2443543: mESC_Rsf1_KO_MNase-seq_rep1; Mus musculus; MNase-Seq
library_construction_protocol: Cell pellets were resuspended in Buffer A (0.25 M sucrose, 60 mM KCl, 10 mM NaCl, 15 mM MES pH 6.5, 5 mM MgCl2, 1 mM CaCl2, 0.5% Triton X-100, 1 mM DTT, 0.2 mM PMSF, 0.5 g/ml leupeptin, 0.7 g/ml pepstatin, and 1 g/ml aprotinin) and incubated on ice for 15 min. Nuclei were collected by centrifugation at 3600 rpm for 10 min and washed once with TNM Buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 2 mM MgCl2, 0.3 M Sucrose, 1 mM DTT, 0.2 mM PMSF, 0.5 g/ml leupeptin, 0.7 g/ml pepstatin, 1 g/ml aprotinin) containing 5 mM CaCl2 (final). 5 l micrococcal nuclease (200 U/l) was added to nuclei suspension and incubated at 37oC for 20 min to give a complete digestion. After incubation, digested nuclei were collected by centrifuging at 3600 rpm for 10 min and resuspended in nucleosome extraction buffer (20 mM Tris HCl pH 7.9, 10 mM EDTA, 0.5 M NaCl). After incubation on ice for 10 min, nuclei were centrifuged at 13,200 rpm for 10 min. The supernatant contains mononucleosomes. Mononucleosomes were treated with 50 μg/ml protease K (Cat. # EO0491, Fisher) at 45°C for 1 hr and then resolved on 1.2% agarose. Gels containing mononucleosomes at the size of 150 bp were excised and purified with Qiagen gel purification kit (Cat# 28704, Qiagen, Waltham, MA, USA). The recovered chromatin DNAs were ligated with 3’- and 5’-adapters for amplification and sequencing by using Illumina II.