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| Status |
Public on May 27, 2016 |
| Title |
RAS2 H4 ChIP-seq |
| Sample type |
SRA |
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| Source name |
IMR90 fibroblasts
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: anti-H4. Millipore, 05-858, rabbit mAb
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| Treatment protocol |
Proliferating IMR90 cells were infected with retroviruses containing empty pBABE-puro vectors (for proliferating control) and pBABE-H-RAS-G12V (to induce OIS). Cells were cultured and passaged for 12 days post infection, while under puromycin drug selection in complete growth medium. Cells were cultured to approximately 95% confluence in 100 mm cell culture dishes containing 10 ml growth medium. This corresponds to approximately 4-5 million proliferating cells or 2 x million senescent cells per 100 mm culture dish. Cells were subjected to cross- linking in the presence of 1% formaldehyde by the addition of 625 μl of 16% formaldehyde (Thermoscientific) to 10 ml of growth medium. Cross-linking was conducted for 10 minutes at room temperature, with gentle agitation on a rocking platform. The cross-linking reaction was quenched for 5 minutes at room temperature by the addition of 500 μl 2.5 M glycine to a final concentration of 125 mM. Cells were detached from the culture dishes using plastic cell scrapers and collected in 50 ml centrifuge tubes kept on ice. Cells were subjected to centrifugation for 5 minutes at 200 x g and 4° C. The supernatant was removed by aspiration, the cell pellets were washed with cold 1X PBS and subjected to centrifugation for an additional 5 minutes at 200 x g and 4° C. The supernatant was removed by aspiration, cell pellets were snap frozen with dry ice and ethanol and stored at -80° C.
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| Growth protocol |
IMR90 cells were cultured in DMEM supplemented with 20% (v/v) FBS, 2 mM L-Glutamine, 50 U/ml Pen-Strep and incubated at 37°C in a humidified 5% CO2 and 3% O2 atmosphere. Every 3-4 days the cells were passaged to maintain 40%–90% confluence level until they stopped proliferating through replicative senescence.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linked cells were pre-extracted with (1:1) modified nuclear lysis buffer (mNLB):IP dilution buffer (IPDB) [35 mM Tris-HCl pH 8.0, 75 mM NaCl, 5.5 mM EDTA pH 8.0, 3 mM EGTA pH 8.0, 0.5% SDS, 0.5% Triton X-100] supplemented with 10 μg/ml aprotinin, 5 μg/ml leupeptin, 50 μg/ml PMSF and sonicated at a density of 2 x 107 cells per 1 ml cold (1:1) mNLB:IPDB plus inhibitors. Sonicated chromatin solutions were cleared by centrifugation, diluted with IPDB [20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS] to a final mNLB:IPDB ratio of 1:10, transferred to microcentrifuge tubes containing antibodies (from Millipore (04-079) and Cell Signaling Technology (5737)) pre-bound to Dynabeads M-280 Sheep anti-Rabbit IgG magnetic beads (Life Technologies), and incubated overnight at 4° C with rotation. The ChIP reactions were washed twice with IPDB, once with High Salt Wash Buffer [20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X- 100], once with LiCl Wash Buffer [10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid] and twice with 1X TE. Beads were aspirated to dryness, resuspended in 500 μl IP Elution Buffer [50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM EDTA pH 8.0, 1% SDS] and 0.5 μl 100 mg/ml RNase A and incubated at 65° C for 4-6 hours. To each tube, 6 μl 20 mg/ml proteinase K was added and the tubes were incubated at 45° C for 12 hours. ChIP DNA was purified usingQIAquick PCR purification kit (50), resuspended with 30 μl buffer (TE) and quantified using the Qubit dsDNA HS Assay Kit and a Qubit fluorometer (Life Technologies).
Libraries were prepared according to NEB's instructions accompanying the NEBNext® Ultra DNA library prep kit for Illumina®(E7370S/L). Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
RAS_H4K20me3_vs_H4_Rep2_FDR01.bed
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| Data processing |
Basecalls performed using CASAVA 1.8.2
Sequenced reads were trimmed using trim galore v0.3.0 with parameter --quality 20 and remaining default settings
Sequenced reads were mapped to hg19 whole genome using bowtie v2.1.0 under default settings
Duplicate reads removed using picard tools v1.98 mark duplicates
Peak finding performed by SICER v1.1 (normalised to histone H4) using redundancy threshold of 1, window size of 200bp, fragment size of 150bp, effective genome fraction of 0.75, gap size of 200bp and R of 0.01.
Peaks from both antibodies were intersected.
Differential peaks were called using Diffbind (v1.8.3)
Genome_build: hg19
Supplementary_files_format_and_content: Peak files (bed format) were generated using SICER, Differential region file (bed format) was generated using DESeq
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| Submission date |
May 27, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Adams |
| Organization name |
University of Glasgow, Beatson Institute for Cancer Research
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| Street address |
Switchback Rd, Bearsden
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| City |
Glasgow |
| ZIP/Postal code |
G61 1BD |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE81969 |
Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability. |
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| Relations |
| BioSample |
SAMN05180631 |
| SRA |
SRX1802450 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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