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| Status |
Public on Feb 12, 2018 |
| Title |
H3K27me3 ChIP-seq Replicate 2 |
| Sample type |
SRA |
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| Source name |
EML_C1
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
passage: 4-5
antibody: H3K27me3, Millipore, cat no: 07-449, lot: 2382150
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| Growth protocol |
EML_C1 was cultured in IMDM medium supplemented with 20% FCS, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 1.5 g/L sodium bicarbonate and 200 ng/ml mSCF.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
H3K27Me3 ChIP samples were fixed using a 1% formaldehyde (FA) fixation protocol for 10 min, while a 60 min, 2 mM DSG and a 60 min 1% FA double fixation protocol was used for Ezh2 and Runx1 antibodies. Fixed chromatin samples were fragmented using a S220 Focused-ultrasonicators (Covaris) for 10 min with 10% duty cycle, 5 intensity, 200 cycle per burst with frequent sweeping mode and constant degassing at 4°C to an average size of 200-500bps. Antibody:chromatin complexes were collected with a mixture of protein A and Protein G Dynabeads (Life Technologies) collected with a magnet, and washed 3X with a solution of 50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate. After a TE wash, samples were eluted, de-crosslinked, RNase and Proteinase K treated, and purified using a QIAGEN PCR purification kit. ChIP samples were quantified relative to inputs. A 50 μl aliquot taken from each of 1 ml of sonicated, diluted chromatin before antibody incubation serves as the input, thus the signal from the input samples represents 5% of the total chromatin used in each ChIP.
The library prep was performed on the Apollo system (Wafergen) with the kit PrepX Complete ILMN 32i DNA Library Kit (cat # 400076) and custom adapters.
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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 |
Reads were aligned with Bowtie2 (version 2.2.1) and the following parameters --maxins 1000 --no-mixed --no-discordant
Duplicate Reads were marked with Picard tools
Duplicate Reads and Secondary alignments were removed with samtools
Ezh2 and Runx1 peaks were called using MACS2 (version 2.0.10) and the following parameters --bdg -g mm --keep-dup 9999
H3K27me3 peaks were called using MACS2 (version 2.0.10) and the following parameters --bdg --broad -g mm --keep-dup 9999
Genome_build: mm10
Supplementary_files_format_and_content: narrowPeak and broadPeak peak files were generated with MACS2
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| Submission date |
Nov 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Adam Mead |
| E-mail(s) |
adam.mead@imm.ox.ac.uk
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| Organization name |
University of Oxford
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| Department |
Weatherall Institute of Molecular Medicine
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| Lab |
HSCB Laboratory
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| Street address |
John Radcliffe Hospital
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| City |
Oxford |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE75186 |
Ezh2 and Runx1 Mutations Targeted to Early Lymphoid Progenitors Collaborate to Promote Early Thymic Progenitor Leukemia [ChIP-Seq] |
| GSE75188 |
Ezh2 and Runx1 Mutations Targeted to Early Lymphoid Progenitors Collaborate to Promote Early Thymic Progenitor Leukemia |
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| Relations |
| BioSample |
SAMN04279041 |
| SRA |
SRX1438365 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1944732_H3K27me3_rep2_peaks.broadPeak.gz |
4.0 Mb |
(ftp)(http) |
BROADPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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