characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-04-01T17:02:53Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-04-11T13:29:20Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA103974418
characteristics.characteristic: External Id
characteristics.text: SickKids Research Institute Department of Molecular Genetics, University of Toronto
characteristics.characteristic: INSDC center name
characteristics.text: 2018-04-01T17:02:53Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-04-11T13:29:20Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1663586
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5670:BRD3-WT2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: U-2 OS cell
characteristics.characteristic: cell line
characteristics.text: 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: Flp-In T-REx U-2 OS cells stably expressing GFP tagged BRD3 WT or a N116A N391A construct (bd(1+2)) were grown in 2 x 15 cm plates to ~75% confluence and induced with 1 µg/mL tetracycline for 48 hours. Cells were then washed with PBS and crosslink with 1% formaldehyde in PBS for 15 minutes at room temperature before quenching the reaction with 125 mM glycine in PBS for 5 minutes at room temperature. Cells were then pelleted and frozen at -80°C until ChIP was performed. Chromatin immunoprecipitation (ChIP) was performed as described previously (REF, PMID:19275939) using 7 µg of anti-GFP (ab290, Abcam) antibody. 220 ng of input control DNA extracted from sonicated cell lysates of each sample were processed in parallel. ChIP DNA was prepared for Illumina sequencing by blunt-end repair, dA-tailing, and ligation of Illumina adaptors using a NEBNext DNA library preparation kit (New England Biolabs, catalogue #E6040L). Total ChIP DNA (approximately 200-500ng) and 220ng of DNA input (WCE) was end repaired for 30 minutes at room temperature, and then purified using column purification with either DNA Clean and Concentrator (Zymogen, catalogue #D4014) or PCR purification columns (Qiagen, catalogue #28106) as recommended by manufacturer’s protocol. Blunt-end repaired DNA was dA-tailed for 40 minutes at 37° C, then column purified. dA-tailed DNA was ligated to Illumina adaptors (final concentration 6.67 nM) that have a T- overhang. USER enzyme was used to cleave the uracil hairpin of the Illumina adaptor, and adaptor-ligated DNA was column purified. The library was PCR amplified for 16-18 cycles using a universal primer and a barcoded primer (New England Biolabs, catalogue #E7335L). PCR-amplified DNA was purified using PCR column purification and eluted in 20 uL of elution buffer for preparation of gel extraction, or 30 uL of TE for preparation of Pippin Prep size selection. Library samples were size selected from 200-350bp using a 2% agarose dye-free automated size selection cassette from Pippin Prep (Sage Sciences, catalogue #CDF2010).
characteristics.characteristic: description
characteristics.text: GFP-BRD3
characteristics.characteristic: genotype
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: BRD3-WT2
characteristics.characteristic: title