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: SAMEA2420690
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: ERS425893
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2368:growing_U2OS_cells_5
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. Bead-bound protein-DNA complexes were washed twice in approximately 14 ml of each wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 150 mM NaCl), wash buffer II (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 500 mM NaCl) wash buffer III (0.25 mM LiCl, 1 % NP-40, 1 % sodium deoxycholate, 1 mM EDTA pH 8.0, 10 mM Tris pH 8.1) and TE buffer (10 mM Tris pH 8.1, 1 mM EDTA pH 8.0) and eluted into 2.5 ml elution buffer containing 1% SDS and 0.1 M sodium bicarbonate for 20 min at 30C while shaking. An equal volume of TE buffer was added to eluates prior to digestion of RNA by incubation with 0.2 mg/ml RNase A (Fermentas) at 37C for 2 hours. Reversal of cross-links was carried out by incubation in the presence of 0.2 M sodium chloride overnight at 65C, followed by incubation with 0.25 mg/mL proteinase K (Roche), 40 mM Tris pH 6.5 and 10 mM EDTA for 1 h at 55C. DNA was purified using a PCR purification kit (Qiagen). To derive genomic DNA to be used as an input reference for SUMO-2 ChIP-seq, a fraction corresponding to the sonicated, diluted and cleared whole cell extracts that had been prepared for SUMO-2 ChIP was removed. These input samples were treated for removal of RNA and cross-link reversal and DNA was extracted as described above. 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 1.8 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 15 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, lysates were incubated with 13 ug SUMO-2 antibody and subsequently with protein G Dynabeads (Life Technologies) for 1 h using an equivalent of 300 ul bead solution. To reduce non-specific binding, beads had been pre-incubated with 0.5% (w/v) BSA in PBS overnight. Bead-bound protein-DNA complexes were washed twice in approximately 13 ml of each wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 150 mM NaCl), wash buffer II (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 500 mM NaCl) wash buffer III (0.25 mM LiCl, 1 % NP-40, 1 % sodium deoxycholate, 1 mM EDTA pH 8.0, 10 mM Tris pH 8.1) and TE buffer (10 mM Tris pH 8.1, 1 mM EDTA pH 8.0) and eluted into 2.3 ml elution buffer containing 1% SDS and 0.1 M sodium bicarbonate for 20 min at 30C while shaking. An equal volume of TE buffer was added to eluates prior to digestion of RNA by incubation with 0.2 mg/ml RNase A (Fermentas) at 37C for 2 hours. Reversal of cross-links was carried out by incubation in the presence of 0.2 M sodium chloride overnight at 65C, followed by incubation with 0.25 mg/mL proteinase K (Roche), 40 mM Tris pH 6.5 and 10 mM EDTA for 1 h at 55C. DNA was purified using a PCR purification kit (Qiagen). 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: 37_2
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_5
characteristics.characteristic: title