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| Status |
Public on Jun 05, 2017 |
| Title |
p65 treated rep2 |
| Sample type |
SRA |
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| Source name |
HepG2
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: NF-κB (Santa Cruz, C-20, sc-372)
cell type: primary epithelial cells
tissue: liver
cell line: HepG2
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| Treatment protocol |
HepG2 cells were incubated at 37°C in 5% CO2, when grown to a density of 1‒5×10^6/mL cells were treated with 10 ng/mL of TNFα in serum-free DMEM at 37 °C for 1 h. Cells were collected by pancreatic enzymes digestion and fixed in vivo with 1% formaldehyde for 10 min at room temperature, followed with 1.25 M glycine for 5 min at room temperature to stop the cross-linking. Then, cells were centrifuged at 1,610g (10 min, 4°C), washed twice in cold PBS containing 1 mM PMSF and 1 mM protease inhibitor cocktail (PIC). Cells were swelled for 20 min on ice in 0.5 mL ChIP cell lysis buffer and centrifuged at 587g (10 min, 4°C). Nuclear pellets were lysed on ice in 0.5 mL ChIP nuclei lysis buffer. The chromatin was sheared by sonication (40×30s on/30s off; Branson, USA) and lysates cleared by centrifugation at 16,100g (15 min, 4°C). Supernatants were collected and stored in aliquots at –80°C for subsequent ChIP. DNA was purified using QIA quick PCR purification kit.
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| Growth protocol |
HepG2 cells were cultured in DMEM medium, complemented with 10% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5‒10 ng of immunoprecipitated DNA were prepared ChIP-Seq libraries.
ChIP-Seq libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Set 2) (New England Biolabs).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
p65_treated.bigWig
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| Data processing |
Sequencing raw data was converted to fastq format and controlled for general quality features using FastQC.
ChIP-seq reads were aligned to unique position on precompiled hg19 reference index by Bowtie with the default setting allowing up to 2 mismatches.
Peaks were identified by MACS v2.1.0 at default settings by comparing specific ChIP-seq reads above with unspecific control reads derived from sequencing matching IgG samples. Peaks were called at a Poisson p-value < 10-4 and an FDR < 5% and were used for intersection analysis to determine the overlap in pair wise comparisons.
All operations on genomic intervals like measuring and counting overlap as well as measuring distances between genomic features were performed using R's GenomicRanges package. In order to identify differentially bound regions all binding regions determined for a given factor under different conditions were collapsed and the read numbers mapping to these collapsed intervals were extracted.
In case the overlap of multiple binding factors was studied at once the multiinter function of the BedTools suite was used with the cluster option turned on.
A peak is assigned to a gene if it locates in the gene region of the gene with HOMER perl script annotatePeaks.pl.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig file
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| Submission date |
May 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Wei Dai |
| E-mail(s) |
[email protected]
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| Phone |
2583793620
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| Organization name |
Southeast University
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| Lab |
State Key Laboratory of Bioelectronics
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| Street address |
Sipailou 2#,Nanjing, Jiangsu Province, P. R. China
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| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
210096 |
| Country |
China |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE98983 |
Genes directly regulated by NF-κB in human hepatocellular carcinoma HepG2 [ChIP-seq] |
| GSE98985 |
Genes directly regulated by NF-κB in human hepatocellular carcinoma HepG2 |
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| Relations |
| BioSample |
SAMN06909706 |
| SRA |
SRX2830428 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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