title: GSM6437283: Histone H3 MNase ChIP-seq total chromatin shRNA O5 Brg1 replicate 2; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin preparation: ES cells were collected and fixed in D15 culture medium containing 1 % formaldehyde (Sigma) for 10 min at 20°C. We stopped fixation by adding glycine to 125 mM, and the cells were centrifugated and washed three times with PBS. Cells were then permeabilized in Tris-HCl pH 7.5 15 mM, NaCl 15 mM, MgCl2 5 mM, EGTA 0.1 mM, KCl 60 mM, Sucrose 0.3 M, 0.4% IGEPAL CA-630 (I3021, Sigma) during 15 min on ice. We next diluted the cells with two volumes of MNase buffer (Tris-HCl pH 7.5 20 mM, NaCl 20 mM, CaCl2 2 mM, MgCl2 4 mM, KCl 15 mM, 0.1 M sucrose), and chromatin was digested during 10 min at 37 °C with 6 Kunitz units of MNase (New England Biolabs, 200 Kunitz units/µl) per 1 million cells. We stopped MNase digestion by adding a final concentration of 10 mM EDTA. We released chromatin from MNase-treated ES cells by passing the cell suspension 13 times through a 26G syringe. Next, we centrifuged the samples, and the supernatants containing the solubilized chromatin were used directly for ChIP-seq experiments or sucrose gradient centrifugation. DNA libraries were prepared using MicroPlex Library Preparation Kit v2 or v3 (Diagenode)