characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-07-16T15:54:27Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:16:58Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2420691
characteristics.characteristic: External Id
characteristics.text: Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, United Kingdom
characteristics.characteristic: INSDC center name
characteristics.text: 2015-07-16T15:54:27Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:16:58Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS425894
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2368:growing_U2OS_cells_4
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: U2OS
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0002869
characteristics.characteristic: cell line
characteristics.text: human osteosarcoma cell
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Human osteosarcoma (U2OS) cells were obtained from CRUK and grown at 37C according to standard cell culture protocols. U2OS cells were grown at 37C and either left untreated or incubated at 43C for 30 minutes to induce heat shock. Approximately 3.5-4.5 x 10 ^ 7 U2OS cells were used per treatment. Cells were chemically cross-linked by adding formaldehyde solution directly into cell culture medium to a final concentration of 1 %. Cells were fixed by incubation at room temperature for 10 min, followed by incubation with 0.125 M glycine for 5 min. Cells were washed twice and scraped into ice-cold PBS. Cell pellets were flash frozen in liquid nitrogen and stored at -80C. Frozen cell pellets were lysed in 2.1 ml lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris (pH 8.1), and protease inhibitors. To shear chromatin to fragments of about 200-500 bp size, samples were sonicated in 300 _l volumes for 14 cycles (7.5 min total sonication time) at high setting using a Bioruptor (Diagenode). Sonicated lysates were then cleared by centrifugation for 10 min at high speed, diluted 1/10 with dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris at pH 8.1) and incubated with SUMO-2 antibody (Life Technologies, 51-9100) and 0.1% Brij-35 overnight at 4C. In detail, to capture protein-DNA complexes, a fraction corresponding to 90% of the lysate volume was incubated with 21 ug SUMO-2 antibody and subsequently with protein G sepharose 4B beads (Sigma) for 1 h using a final bead bed volume of 150 ul. To reduce non-specific binding, beads had been pre-incubated with 0.5% (w/v) BSA in PBS overnight. Libraries were constructed using approximately 10 ng purified DNA. Single-end libraries were generated using either the Illumina ChIP-seq DNA sample Preparation Kit (IP-102-1001) or the TruSeq DNA sample preparation kit (PE-940-2002). In brief, ChIP or input DNA was end repaired and A-tailed before adaptors were ligated on the fragments. The ligated samples were then size selected on a 2 % low melting agarose gel to approximately 250-300 bp. An 18 cycle PCR was performed and libraries were quantified using a Nanodrop Spectrophotometer. Library quality was validated by High Sensitivity Bioanalyzer ChIP (Agilent).
characteristics.characteristic: description
characteristics.text: 43_1
characteristics.characteristic: individual
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: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: growing_U2OS_cells_4
characteristics.characteristic: title