characteristics.text: 2012-10-05T17:01:52Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T15:32:50Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1530213
characteristics.characteristic: External Id
characteristics.text: UNIVERSITY OF MANCHESTER
characteristics.characteristic: INSDC center name
characteristics.text: 2012-10-05T17:01:52Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T15:32:50Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS067617
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-830:ELK1_exp2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: MCF10A
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001200
characteristics.characteristic: cell line
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Nine million MCF10A cells (ATCC CRL-10317) were seeded into 150 mm dishes in media depleted of EGF. After 48 hours cells were incubated for 10 minutes in 1% paraformaldehyde in 1xPBS; the crosslinking reaction was stopped by a 5 minute incubation in 0.125 M glycine in 1xPBS. Cells were washed with ice-cold 1xPBS and harvested into ice-cold 1xPBS containing the Complete protease inhibitor cocktail (Roche, 11836145001). Cells were washed with buffer I (10 mM HEPES pH 6.5, 0.5 mM EGTA, 10 mM EDTA, 0.25% TritonX-100), buffer II (10 mM HEPES pH 6.5, 0.5 mM EGTA, 1 mM EDTA, 200 mM NaCl) and lysed in 370 ul of lysis buffer (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS). Chromatin was sheared by eight 12 second rounds of sonication using Microson XL at power 9. At this stage, a 20 ul portion of the lysate was stored at -20 degrees Celsius until the proteinase K digestion step to be used as an input sample. Next, the total lysate from six plates was diluted 1:10 in IP buffer (16.7 mM TrisHCl pH 8.1, 0.01% SDS, 1.1% TritonX-100, 1.2 mM EDTA, 167 mM NaCl) and incubated overnight at 4 degrees Celsius with 8 ug of an appropriate antibody. For ELK1 (C-term RabMAb, Epitomics 1277-1). For IgG (Normal Rabbit IgG, Millipore, 12-370). Immunocomplexes were then incubated for one hour at 4 degrees Celsius with ProteinG-coated magnetic Dynal beads (Invitrogen, 100.04D) that were pre-blocked overnight at 4 degrees Celsius with sheared salmon sperm DNA (Sigma, D7656-1ML). Beads were washed twice with buffer TSEI (20 mM TrisHCl pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% TritonX-100, 0.1% SDS), once with buffer III (10 mM TrisHCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% IGEPAL, 1% DOC) and twice with buffer TE (10 mM TrisHCl pH 8.0, 1 mM EDTA); all washes were performed for 10 minutes at 4 degrees Celsius. Immunocomplexes were eluted into 150 ul of elution buffer (10 mM NaHCO3, 1% SDS) at room temperature. The input sample was diluted 1:5 with elution buffer. Crosslinks were reversed by addition of NaCl to the final concentration of 250 mM followed by an overnight incubation at 65 degrees Celsius. Protein in samples was digested by a 1 hour incubation with 1 ul of proteinase K (Roche, 031158887001) in 50 mM Tris-HCl pH 6.5 and 8.3 mM EDTA (final concentrations). DNA was purified using the QIAquick PCR purification kit (QIAgene, 28106) and used for sequencing.
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: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: ELK1_exp2
characteristics.characteristic: title