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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 19, 2018 |
| Title |
iRx_H3K79me2_plusDox_ChIPseq |
| Sample type |
SRA |
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| Source name |
induced H3K79me2 ChIPseq
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| Organism |
Mus musculus |
| Characteristics |
strain: 129P2/OlaHsd
cell type: haemogenic endothelium (HE) cells
genotype: induced
antibodies: H3K79me2, Abcam ab3594
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested and washed with PBS before a 2-step crosslinking procedure. First, proteins were crosslinked by incubating cells for 45min at RT in PBS supplemented with 0.83 mg/ml Di(N-succinimidyl) glutarate (DSG, Sigma). Cells were then washed 4 times with PBS, prior to formaldehyde crosslinking of proteins and DNA for 10min at RT using 1% formaldehyde (Pierce) in IMDM with 10% FCS. Formaldehyde was quenched by adding 1/10th volume 2M glycine and crosslinked cells were washed twice in ice-cold PBS. For samples to be used for histone modification ChIP, single crosslinking with 1% formaldehyde was performed. Nuclei were prepared essentially as described in Lefevre et al 2003, sonicated using a Bioruptor water bath in immunoprecipitation buffer I (25 mM Tris 1 M pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% TritonX-100 and 0.25% SDS) {Lefevre, 2003 #45}. After centrifugation the sheared 0.5-2 kb chromatin fragments (1-2 x 106 cells) were diluted with 2 volumes immunoprecipitation buffer II (25 mM Tris pH 8.0, 150mM NaCl, 2 mM EDTA pH 8.0, 1% TritonX-100, 7.5% glycerol). Immunoprecipitation was carried out for 2-4 hours at 4°C using 2 μg antibody coupled to 15 μl Protein-G dynabeads (Invitrogen), with the exception of the H3K79me2 and the H4K5Ac antibodies where immunoprecipitation was carried out overnight. Following IP, the beads were washed with low salt, high salt, LiCl and Na/TE buffers before crosslinks were reversed overnight. DNA was extracted using Ampure beads (Beckman Coulter) and qPCRs were performed to validate ChIP quality.
ChIP-seq libraries were prepared using the Kapa Hyper Prep kit for Illumina platforms according to manufacturer’s instructions
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Quality of reads was verified using FASTQC analysis
The Illumina reads were aligned to the mouse genome using Bowtie2
Reads that were uniquely aligned to chromosomal positions were retained and duplicate reads were removed from the aligned data using Picard tools.
The filtered aligned reads were used to generate density profiles using “genomeCoverageBed” function from bedtools and then converted to big wig file using "wigToBigWig" function
Genome_build: mm10
Supplementary_files_format_and_content: bigWig
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| Submission date |
Sep 20, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Salam Adli Assi |
| E-mail(s) |
s.a.assi@bham.ac.uk
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| Organization name |
University of Birmingham
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| Department |
Institute for Cancer and Genomic Sciences
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| Street address |
IBR
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| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE104045 |
System-wide Dissection of the Transcriptional Response to RUNX1 During Hematopoietic Specification [ChIP-seq] |
| GSE104046 |
The Co-operation of RUNX1 with LDB1, CDK9 and BRD4 Drives Transcription Factor Complex Relocation During Haematopoietic Specification |
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| Relations |
| BioSample |
SAMN07674707 |
| SRA |
SRX3200447 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2788306_iRx_H3K79me2_plusDox_ChIPseq.bw |
203.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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