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| Status |
Public on Nov 26, 2012 |
| Title |
TNFtreated_p65_ChIPSeq |
| Sample type |
SRA |
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| Source name |
HeLa cells
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| Organism |
Homo sapiens |
| Characteristics |
treatment: TNFtreated
cell type: Cervical cancer cell line
chip antibody: p65 (Santa Cruz, C-20, Cat no. SC372)
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| Treatment protocol |
HeLa cells were separated into 3 groups: "control", "TNFα-treated" and "TNFα & MST-312-treated" group. Cells in the "TNFα & MST-312" group were treated with 1 uM of MST-312 (Sigma) for 48 hours prior to the ChIP procedure. Then cells except the control group were treated with TNFα (Calbiochem)to a final concentration of 10 ng/ul for 45mins before the ChIP procedure. The control cells were treated with an equal volume of DMSO (vehicle), for 45mins. For a ChIP experiment, approximately 2 x 10^8 cells from 5 of 150 mm diameter cell culture plates were used.
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| Growth protocol |
HeLa cells were grown to 70-80% confluence in Dulbecco’s modified eagle medium (DMEM) which was supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 2 mM sodium pyruvate and 1 × PSF (penicillin, streptomycin and fungizone) at 37 °C with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% formaldehyde for 10min at room temperature, followed by 125mM glycine treatment to inactivate the crosslinking. Chromatin extracts were sonicated to 200- 500bp fragments, followed by overnight immunoprecipitation at 4˚C.Then, protein-DNA complexes were de-crosslinked with overnight incubation at 65°C and DNA extraction was performed using Qiagen PCR purification kit.
After the quality of the ChIP DNA was controlled by checking the concentration and the enrichment of known targets in ChIP DNA over input with Quant-iT PicoGreen assay (Invitrogen) and real time PCR respectively, libraries were constructed using SOLID ChIP-Seq Kit (Applied Biosystems) following manufacturer's protocol. For each sample, 10 ng of ChIP DNA was purified using AMPure XP Kit (Agencourt), end-repaired and ligated to SOLID adaptors. After ligation, samples were nick-translated and amplified using primers specific to adaptors for 15 cycles. Samples were purified multiple times between each steps. Following the final purification step, size distribution and quantity of libraries were checked by performing DNA 1000 assay (Agilent). Samples with expected size distribution (165-365 bp) and quantity (min 2ng/ul in 10 ul) were sequenced using SOLID4 sequencer following manufacturer's instructions for fragment sequencing. Each sample was loaded to a quarter SOLID slide.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD 4 System |
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| Description |
p65 ChIP-seq, with TNFα
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| Data processing |
35 bp long reads were uniquely mapped to the Human Genome 19 (UCSC), using SOLID Bioscope 1.3.1 ChIP-Seq Module. For alignments, seed and extension approach was used. 30 bp seeds which aligned to the reference genome with at most 3 color mismatches were kept and extended up to 35 bp. Only reads with unique full length alignment (35 bp) allowing for 2 mismatches were used in the analysis.
Redundant reads (multiple reads aligning exactly to the same location) which are generally PCR-artefacts were filtered. Control-based ChIP-seq Analysis Tool version 3 (CCAT3) was performed for peak calling under default settings using reads which were mapped to unique regions in the genome.
Peaks with FDR superior than 0.2 are excluded from the analysis. For the downstream analysis, normalized fold enrichments reported by CCAT3 were used.
Genome_build: hg19 with random chr
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| Submission date |
Sep 24, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Gaye Saginc |
| E-mail(s) |
gayesaginc@gmail.com
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| Organization name |
A*STAR
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| Department |
Genome Institute of Singapore
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| Lab |
Cancer Biology
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| Street address |
60 Biopolis Street
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| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
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| Platform ID |
GPL13393 |
| Series (1) |
| GSE41100 |
Genome-wide TNFα-induced p65 binding before and after telomerase inhibition in HeLa cells |
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| Relations |
| SRA |
SRX189189 |
| BioSample |
SAMN01728935 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1008531_CCAT_CHH907.bed.gz |
20.8 Kb |
(ftp)(http) |
BED |
| GSM1008531_CHH907_mapped.bed.gz |
170.3 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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