Mus musculus
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.
ENA-FIRST-PUBLIC
2018-11-23T17:03:11Z
ENA-LAST-UPDATE
2018-11-13T15:56:02Z
External Id
SAMEA5084581
INSDC center name
WELLCOME TRUST SANGER INSTITUTE
INSDC first public
2018-11-23T17:03:11Z
INSDC last update
2018-11-13T15:56:02Z
INSDC status
public
Submitter Id
E-MTAB-61651542124302:H3K4me1_ChIP_OHT
broker name
ArrayExpress
cell line
E14-ES
cell type
embryonic stem cell
common name
house mouse
disease
normal
genotype
R26::CreERT2; Dpf2flox/flox
growth condition
2D culture
organism part
inner cell mass
sample name
E-MTAB-61651542124302:H3K4me1_ChIP_OHT
scientific_name
Mus musculus