alias: GSM1919544
title: GSM1919544: Mbd3; Mus musculus; MNase-Seq
library_construction_protocol: ES cells were crosslinked with formaldehyde (Sigma) to a final concentration of 1%, incubated at room temperature for 10 min and quenched with glycine to a final concentration of 125 mM. Cells were washed with ice cold PBS and pelleted. Cell pellets were resuspended in lysis buffer (10 mM Tris [pH 7.5], 10 mM NaCl, 2 mM MgCl2, 0.5% NP-40, 1 mM CaCl2) with protease inhibitors (Roche) and incubated for 15 min at 4°C. Permeabilized cells were treated with ~10 Units/106 cells of micrococcal nuclease (Roche) for 5 min at 37°C. Reactions were stopped with the addition of EDTA. Control samples with no MNase addition were treated similarly. Samples were incubated for 4 hr at 4°C with rotation with RNaseA/T1 (Ambion) then incubated overnight at 65°C with 0.01% SDS and proteinase K (Ambion). DNA was extracted with PCI using phase-lock tubes, ethanol precipitated, and resuspended in 30 μL TE. Samples were treated with CIP, end-repaired, A-tailed, and adaptor-ligated as described (Illumina). Between each step, DNA was cleaned with PCI extraction and ethanol precipitation. After adaptor ligation, DNA was selected and purified with Agencourt Ampure beads and then PCR amplified with KAPA HiFi polymerase using 16 cycles of PCR.
identifiers.namespace: GEO
identifiers.id: GSM1919544