GSM1880576: H3K4me3_rep2; Mus musculus; ChIP-Seq
Except for minor modifications to decrease the reaction volume for subsequent ChIP procedure, the chromatin digestion by micrococcal nuclease (MNase) was performed as described by Ozsolak et al (doi:10.1038/nbt1279). E14 mouse embryonic stem cells were harvested and dissociated by Trypsin. Dissociated cells were then resuspended by solution A (300 mM sucrose, 60 mM KCl, 35 mM HEPES pH 7.4, 5 mM K2HPO4, 5 mM MgCl2, 0.5 mM CaCl2) and diluted by solution B (300 mM sucrose, 60 mM KCl, 15 mM NaCl, 35 mM HEPES pH 7.4, 5 mM K2HPO4, 5 mM MgCl2, 3 mM CaCl2) in a 5 millions cells/1.5ml ratio. NP-40 at 0.05% was added to the cell suspension and mixed by pipetting. The lysate of cytoplasm and naked nucleus were checked under microscope. We added either 600U or 1200 U of MNase (New England Biolabs) to the lysate before incubating it at 25◦ C for 15 or 45 min. After this, the MNase digestion was slowed down by putting the lysate on ice, and the nucleus were collected by centrifugation at 3000 rcf at 4◦ C for 10 min. We re-suspended the nucleus on solution C (100 mM EDATA 4% SDS) in a 5 millions cell/150μL ratio. Then, we added 100 mL of solution C to stop the MNase digestion and lysate the nucleus membranes. The lysates were treated with RNase and proteinase K, and 1/5 of it was kept for DNA purification. Nucleosomal DNA was purified by phenol chloroform and precipitated by isopropanol. MNase-digested DNA coupled with ChIP-seq  (MNChIP-seq)
GEO Accession
GSM1880576