characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-10-20T17:09:35Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-10-20T17:09:35Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112156048
characteristics.characteristic: External Id
characteristics.text: Southern University of Science And Technology
characteristics.characteristic: INSDC center name
characteristics.text: 2023-10-20T17:09:35Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-10-20T17:09:35Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14265945
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12390:mESC_Con1_H3K27me3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: ES-E14
characteristics.characteristic: cell_line
characteristics.text: embryonic stem cell
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: 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. Mouse E14 ESCs were cultured in DMEM (Hyclone) supplemented with 10% FCS (Gibco), 1xNEAA (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 2 mM L-glutamine (Gibco), and LIF (Millipore) on 0.1% gelatin (Sigma) coated plates. 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 the Illumina Nextera DNA Library Preparation Kit.
characteristics.characteristic: description
characteristics.text: embryo
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: none
characteristics.characteristic: environmental stress
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: embryoid body
characteristics.characteristic: growth condition
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: inner cell mass
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: 129/Ola
characteristics.characteristic: strain
characteristics.text: mESC_Con1_H3K27me3
characteristics.characteristic: title