characteristics.text: 2012-11-01T17:02:06Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:00:00Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1559032
characteristics.characteristic: External Id
characteristics.text: GIS
characteristics.characteristic: INSDC center name
characteristics.text: 2012-11-01T17:02:06Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:00:00Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS177700
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1305:EZH2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: H1-hESC
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0003042
characteristics.characteristic: cell line
characteristics.text: embryonic stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002322
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: ChIP-SeqH1 hESCs were maintained as feeder-free culture on matri-gel (BD). The medium containing 20% KO serum replacement, 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM 2-mercaptoethanol and 4ng/ml basic fibroblast growth factor (Invitrogen) in DMEM/F12 (GIBCO) was conditioned with mitotically inactivated mouse embryonic fibroblast (MEF) for 24hrs. Additional 8ng/ml of basic fibroblast growth factor (Invitrogen) was supplemented to conditioned medium before use. Medium was changed daily. The hESCs were passaged with 1 mg/ml collagenase IV (Gibco) upon confluency.ChIP-Seq ChIP-SeqThe EZH2 antibody used in the ChIP is from Active motif (Cat: 39901). Between 10e7-10e8 cells were used for the ChIP experiment. Crosslinking of protein and DNA was done at room temperature for 10 minutes with serum free media that contains 1% formaldehyde. The crosslinking reaction is quenched with 0.2M final concentration of glycine for 5 minutes. Cells were washed extensively with 1XPBS, scraped and pelleted. The cells were lysed with 0.1% SDS buffer and collected nuclei were lysed with 1% SDS buffer. Pelleted chromatin was resuspended in the 0.1% SDS buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 1 mM EDTA) and the DNA was sheared via sonication with the BioRuptor (Diagenode) for 16 cycles of 30 seconds with 1 min intervals at amplitude 40. The chromatin sample was precleared at 20,000rpm for 45mins before the eluents are used for ChIP. ChIP was performed with 5ug of the antibody using Magnetic Protein A beads (Invitrogen). The chromatin was incubated with the immobilized antibody overnight. The ChIP sample was washed 3 times with the IP (0.1%SDS) buffer, once with 1M NaCl in the same buffer, once with LiCl buffer and once with TE-buffer and DNA-protein complexes were eluted with 50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% SDS at 69C for 30mins in a orbital shaker at 14000 rpm. The protein-DNA complex was dissociated with 1.5ug/ml Pronase at 42C for 2 hr followed by 67C for 6 hr. ChIP DNA from 2 duplicates were pooled for 10ng of starting material for the library construction. The ChIP enriched DNA was modified with the ChIP-Seq DNA Sample Prep Kit (IP-102-1001, Illumina). Briefly, the DNA end was repaired with exonucleases and an adaptor was ligated to the end, followed by PCR amplification for 15 rounds. The amplified DNA was gel purified and the 200-300bp fragments were selected for subsequent SOLEXA sequencing (Illumina) using the Illumina 1G instrument according to the manufacturers protocol for 36 bp reads.ChIP-Seq
characteristics.characteristic: description
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: EZH2
characteristics.characteristic: title