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| Status |
Public on Jun 22, 2019 |
| Title |
CSB+H3K9me3 ChIP for rK9 |
| Sample type |
SRA |
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| Source name |
Patient cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Fibroblast
cell line description: CS1AN cells that stably expressing wild type CSB that used for rK9 peak calling
chip antibody: anti-H3K9me3, Millipore 07-442
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| Treatment protocol |
2.5 mM H2O2 for 30minutes, 10 or 50 ng/ml of doxycycline for 72 hours
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| Growth protocol |
SV40-transformed CS1AN cells stably transfected with either CSB or empty vectors were cultured in DMEM supplemented with 10% FBS, 1% pen-strep, and 400 µg/ml geneticin and grown in 20% O2/5% CO2 at 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CSB cells were cross-linked in a solution of 1% formaldehyde in PBS for 10 min at room temperature. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M. The cells were harvested and washed three times with cold PBS, and cytosolic fractions were eliminated with buffer A (5 mM PIPES (pH 8.0), 85 mM KCl, 0.5% NP-40, protease inhibitor cocktail (GenDEPOT, Katy, TX, USA)). Nuclear pellets were resuspended in buffer B (100 mM Tris-Cl (pH 8.1), 1% sodium dodecyl sulfate (SDS), 10 mM EDTA, protease inhibitor cocktail), and the chromatin was sheared with S-450 sonicator (Branson, Danbury, CT, USA). Sonication condition was optimized by analyzing purified DNA samples with BIOANALYZER (Agilent, DNA1000 Kit)The prepared chromatin fraction (500 μg total sheared DNA per sample) was diluted 1/10 in IP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl (pH 8.1), 167 mM NaCl and a protease inhibitor cocktail) and incubated with antibodies, overnight at 4 °C. Samples were incubated for 2–4 h at 4 °C with protein A or G beads. Then the beads were washed with TSE150 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl (pH 8.1), 150 mM NaCl), TSE500 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl (pH 8.1), 500 mM NaCl), Buffer III (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-Cl (pH 8.1)) and two times with TE (pH 8.0) for 10 min in each solution. Bead-bound chromatin was eluted with elution buffer (1% SDS, 0.1 M NaHCO3 (pH 8.0)) for 1-2 hours at 65.5 °C. The supernatant containing the chromatin, without the beads, was isolated and incubated overnight at 65 °C with 200 mM NaCl to reverse cross-linking. Five hundred microliters of the sample were incubated at 50 °C after adding 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris (pH 6.5) and 4 μl of Proteinase K (20 mg ml−1) and then purified with phenol/chloroform/isoamyl alcohol. Nucleic acids were precipitated by centrifugation for 30 min at 4 °C after mixing the sample with 1 μl of glycogen solution (20 mg ml−1), 20 μl 5 M NaCl and 500 μl isopropanol. Purified nucleic acid pellets were washed with 70% ethanol, dried and dissolved in nuclease-free water.
Library construction according to the manufacturer's protocol
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
H3K9me3 ChIP
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| Data processing |
The sequencing data were uploaded to the Galaxy web platform, and we used the public server at usegalaxy.org to analyze the data.
Sequencing reads were mapped against the human genome (GRCh37/hg19) using Bowtie for Illumina with default parameters
The SAM format outputs were sorted by genomic coordinates and uniquely mapped reliable reads were used in subsequent steps. SAM files were preprocessed using SAMtools
We used the MACS tool to select regions that were enriched for H3K9me3 and PAR. We applied the default settings and found significant regions (P-value ≤ 10-5), using non-IP chromatin as a control to eliminate unspecific signals.
Genome_build: hg19
Supplementary_files_format_and_content: bed files with called peaks, bw files with coverage
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| Submission date |
Jun 21, 2019 |
| Last update date |
Jun 26, 2019 |
| Contact name |
Jong-Hyuk Lee |
| E-mail(s) |
[email protected]
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| Organization name |
National Institute of Health
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| Lab |
Laboratory of Molecular Gerontology
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| Street address |
251 Bayview BLVD
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| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE133176 |
Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging |
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| Relations |
| BioSample |
SAMN12108880 |
| SRA |
SRX6183290 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3901419_rK9.interval.txt.gz |
268.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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