characteristics.text: 2014-12-12T09:23:58Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:11:31Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2043666
characteristics.characteristic: External Id
characteristics.text: University of Cambridge
characteristics.characteristic: INSDC center name
characteristics.text: 2014-12-12T09:23:58Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:11:31Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS206786
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1443:BRD4_LMB
characteristics.characteristic: Submitter Id
characteristics.text: 2 month
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UO_0000035
characteristics.characteristic: age
characteristics.text: frozen_sample
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000922
characteristics.characteristic: bio source type
characteristics.text: ATCC
characteristics.characteristic: biosourceprovider
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: OCIAML3
characteristics.characteristic: cell line
characteristics.text: myelomonocyte
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_1000309
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: OCIAML3 cells were grown in RPMI-1640 medium (Sigma-Aldrich) supplemented with 80% alpha-MEM + 20% fetal calf serum and 1% penicillin/streptomycin. Cells were incubated at 37C and 5% CO2. OCIAML3 cells were treated for 6 h with either I-BET151, leptomycin B (LMB) or DMSO (vehicle) alone. From these cells, chromatin was prepared and chromatin immunoprecipitation was performed using an anti-BRD4 antibody (ab75898, Abcam). Cells were crosslinked with 1% (v/v) formaldehyde for 15 min at room temperature; crosslinking was stopped by the addition of 0.125 M glycine. Cells were then lysed in 1% (w/v) SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0, 1 mM sodium orthovanadate and protease inhibitors. Cells were sonicated in a Bioruptor (Diagenode) to achieve a mean DNA fragment size of 500 base pairs. An equal volume of Protein-A and Protein-G agarose beads, pre-absorbed with sonicated salmon-sperm DNA and BSA, were used to preclear the chromatin for 2 h before immunoprecipitation. Immunoprecipitation was performed for a minimum of 12 h at 4C in modified RIPA buffer (1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 90 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM sodium orthovanadate, EDTA-free protease inhibitors). An equal volume of Protein-A and Protein-G agarose beads, pre-absorbed with sonicated salmon-sperm DNA and BSA, were used to bind the antibody and associated chromatin. The beads were washed before elution of the antibody-bound chromatin. Reverse crosslinking of DNA was followed by DNA purification with the QIAquick PCR purification kit (Qiagen). Immunoprecipitated DNA was amplified for sequencing using TaqMan PCR mastermix according to the manufacturers instructions. ChIP libraries used for massive parallel sequencing were prepared using the Illumina ChIP-seq kit with the following adjustments. Following chromatin immunoprecipitation and DNA purification, 10-50ng of DNA was used to perform end repair of the ChIP DNA. The following reaction mix was used, DNA (10-50ng resuspended in 30 ml), DNase and RNase free ddH2O 10 ml, T4 DNA ligase buffer with 10 mM ATP 5 ml, 10 mM dNTP mix 2 ml, T4 DNA polymerase 1 ml, Klenow DNA polymerase 1 ml and T4 PNK 1 ml. The sample was incubated for 30 min at 20 C and then purified using the Zymo DNA Clean & Concentrator-5O kit with the DNA eluted in 11 ml of ddH2O. Following end repair adenine bases were added to the 30 end of the DNA fragments using DNA from above 11 ml, ddH2O 33 ml, Klenow buffer 5 ml, dATP 10 ml and Klenow (30 to 50 exonuclease deficient) 1 ml. The sample was incubated for 30 min at 37 C and the DNA was purified using the Zymo kit. Commercially supplied adapters (Illumina; the sequence is commercially protected) were then ligated to the DNA fragments using DNA ligase buffer 14 ml, adaptor oligonucleotide mix 2 ml, DNA ligase 4 ml and DNA 10 ml. The reaction was incubated for 15 min at room temperature and DNA purified using the Zymo kit. Following the ligation of adapters the DNA was amplified by PCR. The reaction conditions included DNA 10 ml, ddH2O 26 ml, 5X Phusion buffer 10 ml, 10 mM dNTP mix 1.5 ml, Phusion polymerase 0.5 ml and 1 ml of both forward and reverse PCR primers the sequence of which is commercially protected. The reaction conditions are as follows: 98 C, 30 s; 98 C, 10 s; 65 C, 30 s; 72 C, 30 s; back to 2 (18 cycles); 72 C for 5 min. Following PCR amplification, the DNA was purified using the MinElute PCR purification kit (QIAGEN). The clean DNA was then size fractionated in a 1.5% agarose gel. Each sample was run in isolation in the gel in a dedicated clean agarose tank in 1 X TAE buffer. The gel was subsequently stained with SybrGreen in a clean staining tank for 1 hr. Following this, 200 -400 bp fragments were excised from the DNA smear present on the gel, and purified using the MinElute QIAGEN Gel-Purification Kit. The completed library was then tested for both size selection and quantity of DNA using a 2100 Agilent Bioanalyser.
characteristics.characteristic: description
characteristics.text: acute myeloid leukemia
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000222
characteristics.characteristic: disease
characteristics.text: ATCC_OCIAML3
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: blood
characteristics.characteristic: organism part
characteristics.text: NPM1c AML
characteristics.characteristic: phenotype
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: BRD4_LMB
characteristics.characteristic: title