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| Status |
Public on Sep 20, 2015 |
| Title |
E14Tg2a Ring1B ChIP rep2 |
| Sample type |
SRA |
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| Source name |
E14Tg2a mESCs
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| Organism |
Mus musculus |
| Characteristics |
gender: male
strain: 129P2/OlaHsd
cell type: Embryonic Stem Cells
antibody: Ring1B (MBL D139-3)
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| Growth protocol |
ES cells were grown in GMEM supplemented with 10 % fetal bovine serum (FBS), 1,000 units/ml LIF, non-essential amino acids, sodium pyruvate, 2-β-mercaptoethanol, L-glutamine and Penicillin/Streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Trypsinised ESCs (20 million) were washed twice in PBS. Cells were resuspended in 250ml of PBS and fixed by the addition of an equal volume of PBS containing 2% methanol free formaldehyde (Thermo Scientific Pierce PN28906; final concentration of 1%) and incubated at RT for 10 mins. Fixation was stopped by 5 min incubation with 125mM glycine at room temperature. Cells were washed in PBS prior to lysis. All of the buffers were supplemented with the following additives just prior to use: 0.2 mM PMSF, 1 mM DTT, 1x Protease inhibitors (Calbiochem, 539134-1SET). Cell pellets were resuspended in lysis buffer 1 (50mM Tris-HCl pH 8.1, 10mM EDTA and 20% SDS) and incubated for 10 min at 4°C. Lysates were diluted 1:10 in ChIP dilution buffer (0.1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM and Tris-HCl pH8.1) and sonicated using a chilled Bioruptor (Diagenode; 40x 1 min cycles of 30sec on / 30sec off on ‘high’ setting at 4°C). The sonicated extract was pre-cleared by centrifugation at 16000g for 10 min at 4°C. The supernatant was transferred to a fresh tube and supplemented with BSA and triton X-100 to a final concentration of 25mg/ml and 1% respectively. A sample of the chromatin was retained as an input reference. Antibodies were pre-coupled to a 1:1 mix of protein A and G Dynabeads (Life Technologies; 10001D and 10004D respectively) at a ratio of 1mg antibody per 30ml of dynabeads. 12 million and 6 million cell equivalents of lysate were added to 10ug of Anti-Ring1B (MBL D139-3) or 5ug anti-H3K27me3 (Millipore 07-449) respectively and incubated for 10hrs on a rotating wheel at 4ºC. Bead-associated immune complexes were washed sequentially with wash buffers A, B and C for 10 mins at 4ºC on a rotating wheel followed by 2 washes in TE buffer at RT (wash buffer A - 1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM and Tris-HCl pH8.1; wash buffer B - 1% Triton X-100, 0.1% Sodium-Deoxycolate, 0.1% SDS, 1mM EDTA, 500mM NaCl, 20mM and Tris-HCl pH8.1; wash buffer C – 1% NP40, 0.1% Sodium-Deoxycolate, 1mM EDTA, 250mM LiCl, 20mM and Tris-HCl pH8.1). Chromatin was released from the beads by incubation with elution buffer (0.1 M NaHCO3 and 1 % SDS) for 15 mins at 37 ºC followed by the addition of RNaseA and Tris pH6.8 (final concentration of 20mg/ml and 100mM respectively) and incubation at 65ºC for 2 hours following by the addition of 50mg of proteinase K and incubation at 65ºC overnight to degrade proteins and reverse the cross-links. Dynabeads were removed using a magnetic rack and the chromatin purified using PCR Purification columns (Qiagen) according to manufacturer’s instructions.
libraries were prepared as previously described (Bowman et al., 2013 - PMID:23837789) with the following modifications. No purification was performed between the A-tailing reaction and the ligation reactions; instead the ligation was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5% PEG-6000, 1mM ATP and 13.3nM of annealed Illumina adaptors (AU) and incubated at 25°C for 90min. Size selection purifications following the ligation and amplification PCR steps were performed with 1x and 0.8x reaction volumes of Agencourt AMPure XP beads (Beckman Coulter - A63880). Six samples were multiplexed per Hi-seq lane.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Ring1B ChIP in E14Tg2a mouse ESCs
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| Data processing |
Base calling was performed using casava (version 1.8.2).
Reads were trimmed with Trim Galore! v0.2.7. The -q 30 option was used to remove reads with a PHRED score lower than 30 using cutadapt v1.2.1.
Trimmed reads were mapped to the mouse genome (mm10) using bowtie2.1.0 with the following arguments: --local -D 20 -R 3 -N 1 -L 20 -i S,1,0.50.
The obtained SAM files were processed using HOMER v4.3. HOMER tag directories were created using the makeTagDirectory tool with the options -unique and -fragLength 150. These tag directories were used to create bedgraphs for UCSC visualisation (bedGraphs); for identification of enriched regions (Homer peak files); and for quantifications shown in the manuscript. Bedgraphs were created using the makeUCSCfile tool with default options. Enriched regions were identified for ChIP-seq samples using the findPeaks tool with the options -style histone and -minDist 500 with the input sequences as controls.
Sites of robust Ring1b and H3K27me3 enrichment were identified as Homer peaks which were consitently identified in both biological replicate ChIPs (included as supplementary files - Strict peak file)
Genome_build: mm10
Supplementary_files_format_and_content: Bedgraphs were made using HOMER tools and allow the mapped reads to be visualised in a genome browser. Peak files contain the output of the findPeaks tool from HOMER tools. The set of strict peaks (supplementary files) was produced by finding regions of at least 500 bp which are identified as peaks in each replicate of an experiment.
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| Submission date |
Jun 17, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Rob S Illingworth |
| E-mail(s) |
robert.illingworth@ed.ac.uk
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| Phone |
01316519640
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| Organization name |
The University of Edinburgh
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| Department |
Centre for regenerative Medicine
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| Lab |
Illingworth
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| Street address |
Centre for Regenerative Medicine
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| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH16 4UU |
| Country |
United Kingdom |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE69955 |
Mouse ES cells expressing catalytically inactive Ring1B display impaired Ring1B and H3K27me3 deposition. |
| GSE69978 |
Loss of Ring1B catalytic activity causes a pronounced reduction in H3K27me3 deposition yet minimally disrupts the expression of target genes |
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| Relations |
| BioSample |
SAMN03779371 |
| SRA |
SRX1063117 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1713907_mm10_wt_ring1b_2_homer_regions.txt.gz |
427.2 Kb |
(ftp)(http) |
TXT |
| GSM1713907_mm10_wt_ring1b_2_ucsc.bedgraph.gz |
77.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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