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| Status |
Public on Apr 04, 2016 |
| Title |
H3K18ac.SIRT6KD_rep2 |
| Sample type |
SRA |
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| Source name |
U2Os cells
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| Organism |
Homo sapiens |
| Characteristics |
cell: U2Os cells
treatment: SIRT6 Knock Down
antibody: anti H3K18Ac abcam ab1191
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| Treatment protocol |
For lentiviral packaging, 293T cells were co-transfected with pVPack-VSV-G, Δ8.2 and pSicoR containing shRNAs directed against SIRT6 or control constructs, and viral supernatant was collected after 48 h. For transduction, cells were incubated with virus-containing supernatant in the presence of 8 μg/ml polybrene. After 48 h, infected cells were selected for 72 h with puromycin (1.5 μg/ml). SIRT6 knockdown target sequence: S6KD 5’ -AAGAATGTGCCAAGTGTAAGA- 3′
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| Growth protocol |
U2OS cells were growth in MEM with 10% FBS, 2mM L-Glutamine and Pen/Strep
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 8 min, quenched with 1.25 M glycine, washed in PBS. Cells were resuspended in cells lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, 1x Roche Complete Protease inhibitor), kept in ice for 10 min, and sonicated using Bioruptor. Following sonication insoluble material was pelleted by centrifugation at 16000g for 10 minutes at 4°C. A small sample of soluble crosslinked chromatin was purified, checked for correct sonication size range. 25 ug of chromatin was diluted to 350ul with RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate, 1x Roche Complete Protease inhibitor). 10 μl of Protein A Dynabeads (Invitrogen) prebound with 2 μg of histone marks antibodies were added. Samples were incubated overnight at 4°C with rotation. Beads were precipitated on magnet stand and washed 3 times with 1 mL of RIPA buffer and once with TE. Crosslink was reversed with 250 μl of elution buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 5 μg/mL proteinase K) at 65°C for 2 hours. DNA was purified with Qiagen PCR cleanup kit. Concentration of eluted chromatin was measured with Qubit dsDNA HS Assay Kit (Life Technology).
Sequencing libraries were prepared using the Illumina TruSeq DNA Sample Preparation Kit according to the manufacturer's protocol. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250-450 bp (insert plus adaptor) were band isolated from an agarose gel. DNA fragments were sequenced using paired-end sequencing technology on Illumina HiSeq 2000 platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
anti H3K18Ac abcam ab1191
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| Data processing |
Basecalls performed by Illumina CASAVA 1.8.2
ChIP-seq reads were aligned to the hg19 genome using Bowtie2 v2.1.0
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq wig files were generated using DANPOS 2; Scores represent the ChIP-seq tag numbers
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| Submission date |
Jun 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuanxin Xi |
| E-mail(s) |
xiyuanxin@yahoo.com
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| Phone |
530-220-2067
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| Organization name |
The University of Texas MD Anderson Cancer Center
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| Department |
Bioinformatics and Computational Biology
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| Street address |
1400 Pressler St
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE69809 |
Silencing of pericentric heterochromatin associated with SIRT6-dependent H3K18 deacetylation protects against mitotic errors and cellular senescence |
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| Relations |
| BioSample |
SAMN03771149 |
| SRA |
SRX1057274 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1709277_H3K18ac.SIRT6KD_rep2.wig.gz |
350.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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