title: p300_ChIP_WT
organism: Mus musculus
description: Protocols: Cells were grown to a final concentration of 5x107 cells for each ChIP-seq experiment. To stabilize Dpf2 on chromatin, cells were treated with 2 mM disuccinimidyl glutarate (DSG) for 10 minutes prior to formaldehyde crosslinking. For all other targets, cells were chemically cross-linked at room temperature by the addition of formaldehyde to 1% final concentration for 10 minutes and quenched with 0.125 M final concentration of glycine. Cross-linked cells were re-suspended in sonication buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate, 0.1% SDS) and sonicated using a Diagenode Bioruptor for three 10-minute rounds using pulsing settings (30 sec ON; 1 min OFF). 10 µg of sonicated chromatin was then incubated overnight at 4ºC with 5 µg of Flag antibody conjugated to magnetic beads. Following the IP, beads were washed twice with RIPA buffer (50mM Tris-HCl pH8, 150 mM NaCl, 2mM EDTA, 1% NP-40, 0.1% Na-deocycholate, 0.1% SDS), low salt buffer (20mM Tris pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20mM Tris pH 8.1, 500mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10mM Tris pH 8.1, 250mM LiCl, 1mM EDTA, 1% Na-deoxycholate, 1% NP-40), and 1X TE. Finally, DNA was extracted by reverse crosslinking at 65ºC overnight with proteinase K (20ug/µL) and 1% SDS followed by phenol:chloroform:isoamyl alcohol purification and ethanol precipitation. Libraries were constructed as indicated above and sequenced using Illumina HiSeq 2500. ChIPed DNA was purified using Qiagen MinElute PCR Purification Kit. Library DNA was purified using AmpureXP beads using 1:1 ratio. Libraries were constructed using Diagenode MicroPlex ChIP kit.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-11-23T17:03:11Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-11-13T15:56:02Z
attributes.tag: External Id
attributes.value: SAMEA5084588
attributes.tag: INSDC center name
attributes.value: WELLCOME TRUST SANGER INSTITUTE
attributes.tag: INSDC first public
attributes.value: 2018-11-23T17:03:11Z
attributes.tag: INSDC last update
attributes.value: 2018-11-13T15:56:02Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-61651542124302:p300_ChIP_WT
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: E14-ES
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: 2D culture
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: sample name
attributes.value: E-MTAB-61651542124302:p300_ChIP_WT
attributes.tag: scientific_name
attributes.value: Mus musculus