U.S. flag

An official website of the United States government

Format

Send to:

Choose Destination

SRX6183272: GSM3901400: CSB-DMSO H3K9me3 input; Homo sapiens; ChIP-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 43.1M spots, 2.9G bases, 1.2Gb downloads

Submitted by: NCBI (GEO)
Study: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging
show Abstracthide Abstract
Cockayne syndrome (CS) is an accelerated aging disorder, caused by mutations in the CSA or CSB genes. In CSB-deficient cells, poly (ADP ribose) polymerase (PARP) is persistently activated by unrepaired DNA damage and PARP consumes and depletes cellular nicotinamide adenine dinucleotide (NAD), which leads to mitochondrial dysfunction. Here, the distribution of poly (ADP ribose) (PAR) was determined in CSB-deficient cells using ADPr-ChAP (ADP ribose-chromatin affinity purification), and the results show striking enrichment of PAR at transcription start sites (TSS), depletion of heterochromatin, and downregulation of H3K9me3-specific methyltransferases SUV39H1 and SETDB1. Induced-expression of SETDB1 in CSB-deficient cells downregulated PAR and normalized mitochondrial function. The results suggest that defects in CSB are strongly associated with loss of heterochromatin, downregulation of SETDB1, increased PAR in highly-transcribed regions, and mitochondrial dysfunction. Overall design: H3K9me3 ChIP or ADPr-ChAP was performed from CSB-deficient (CS1AN) /control cells (CS1AN expressing wild type CSB) or doxycyclin-induced SETDB1 expressing CSB-deficient cells.
Sample: CSB-DMSO H3K9me3 input
SAMN12108869 • SRS5004019 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction 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. According to the manufacturer's protocol
Experiment attributes:
GEO Accession: GSM3901400
Links:
Runs: 1 run, 43.1M spots, 2.9G bases, 1.2Gb
Run# of Spots# of BasesSizePublished
SRR941742543,067,5042.9G1.2Gb2019-06-27

ID:
8237719

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...