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| Status |
Public on Dec 11, 2015 |
| Title |
HPT.Blue.ChIPSeq.R2.ChIP |
| Sample type |
SRA |
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| Source name |
HEK293 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic semi-differentiated kidney cells
sequencing time: 2
fraction: mock IP
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| Treatment protocol |
To induce the production of the HPT-BAHD1 fusion, tetracycline was added to cell culture media 30 h before cell recovery at a final concentration of 11 µg/ml. Tetracyclin was also added to the cultures of the control HEK293-HPT-blue cells.
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| Growth protocol |
Stable HEK293-HPT-BAHD1 and control HEK293-HPT-blue cells were generated as described (Lebreton et al. Science 2011, PMID: 21252314). Stable pool of transfectants having undergone integration of HPT-expressing plasmids at the single FRT locus were grown under 5% CO2 atmosphere, in DMEM supplemented with 10% (v/v) Fetal Calf Serum (FCS) and Penicillin/Streptomycin (0.1 mg/ml each), 100 mg/ml hygromycin to maintain the integration and 15 μg/ml of Blasticidin in order to select for the maintenance of Tet repressor expression. Cellular amplification was performed in 2-liter spinner flasks (3 flasks containing 1L of the same medium per condition and replicate) as thoroughly described (Derivery et al. Methods Enzymol. 2010, PMID: 21036256) for one week.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Preparation of solubilized chromatin fractions from these cells (Fritsch et al. Mol. Cell 2010, PMID: 20129054) and tandem affinity purification (Lebreton et al. Science 2011, PMID: 21252314) were performed as previously described. Only the eluates from the first affinity column were used for DNA extraction. DNA was purified by two extractions with equal volumes of phenol:chloroform:isoamy lalcohol (25:24:1, pH=8), starting from 10 µL of the input samples diluted in 200 µL water, or 200 µL of eluates. Phenol was eliminated with one extraction with chloroform, then DNA was precipitated by addition of 900 µL of ethanol, 100 µL of ammonium acetate 7.5 M and 0.2 µL of glycogen (20 µg/µL) to each aqueous phase, incubation at -80°C for 30 min and centrifugation at 4°C, 20,000 ´ g for 15 min. The pellet was washed once in 100 µL of ethanol, then resuspended in 50 µL of pure water for inputs, 25 µL for eluates. 2 independent experiments were performed (R1 and R2)
The library construction was performed by Fasteris according to the Illumina guidelines, with the following steps: 3’ A addition; Ligation of adapters; Gel purification to isolate fragments of specified inserts size; PCR amplification to generate the DNA Colonies Template Library, library purification; Library quantification and dilution at 10 nM. Library Quality Control, done by titration run on the HiSeq. Independent libraries were produced for R1 and R2. The DNA Colony Template library was sequenced as single reads of 50 bp. Sequencing was multiplexed at 3 libraries per channel. For the HPT-BAHD1 ChIP sample, the R2 library was sequenced twice (R2.a and R2.b).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
control
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| Data processing |
Basecalls performed using CASAVA version 1.8
reads were filtered with custom Python scripts
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie with default parameters, clonal reads were removed
enriched regions were selected with CCAT (v3.0) using individual replicates of ChIP vs Input with the following parameters: fragmentSize =150, slidingWinSize= 1000, movingStep=50, isStrandSensitiveMode = 0, minCount =4 (R1), 8 (R2), outputNum =10000, randomSeed = 123456, minScore =5.0 (R1) (fold change criteria) bootstrapPass =50.
reads were assigned to genomic windows of 400 bp using Repitools
counts were normalized with edgeR per effective library size
regions selected with CCAT were used to subset the table of library size normalized counts per genomic window
a generalized linear model was run with edgeR with time and IP as confounding factors
Genome_build: hg19 (female)
Supplementary_files_format_and_content: bedGraph files, library size normalized counts per 400 bp genomic window
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| Submission date |
Dec 16, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Helene Bierne |
| E-mail(s) |
helene.bierne@inrae.fr
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| Phone |
33134652289
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| Organization name |
INRAE - University PARIS SACLAY
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| Department |
Micalis Institute
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| Lab |
Epigenetics and Cellular Microbiology
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| Street address |
Domaine de Vilvert, bat 442, INRA Micalis
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| City |
Jouy-en-Josas |
| ZIP/Postal code |
78350 |
| Country |
France |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE51868 |
HEK293 cells (HEK293-CT) and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1) |
| GSE53372 |
Chromatin immunoprecipitation-sequencing of HEK293 cells (HEK293-HPT-blue) and HEK293 cells stably over-expressing the BAHD1 gene (HEK293-HPT-BAHD1) |
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| Relations |
| BioSample |
SAMN02444976 |
| SRA |
SRX392838 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1290247_HPT.Blue.R2.library.norm.bedGraph.gz |
74.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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