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| Status |
Public on Mar 03, 2017 |
| Title |
Input-seq P19, RA 24hrs |
| Sample type |
SRA |
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| Source name |
RA-treated P19 EC cells
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| Organism |
Mus musculus |
| Characteristics |
cell-type: P19 embryonal carcinoma (EC) cells treated 24 hrs with RA
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| Growth protocol |
P19 EC cells were routinely grown in DMEM complemented with 10% fetal calf serum. P19 EC cells were treated with 10-6 M retinoic acid for 0, 6, 12, 18, 24 or 48 hrs. Cells were either collected without crosslinking for genomic DNA extraction or fixed with 1.5% formaldehyde for chromatin immunoprecipitation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For MeDIP and hMeDIP, cells were scraped in phosphate buffered saline and were pelleted at 100 g before genomic DNA extraction using a DNeasy Blood and Tissue kit (Qiagen, France). For ChIP, P19.6 cells were cross-linked with 1.5% formaldehyde for 10 min at room temperature. The cells were rinsed with cold PBS, harvested and lysed with 1% SDS, 10 mM EDTA, and 50 mM Tris-HCl (pH 8.1) containing a protease inhibitor cocktail (Roche). Chromatin fragmentation was subsequently obtained by sonicating samples for 14 min (30 sec on/off cycles) using a Bioruptor (Diagenode) set up at the highest intensity. The soluble chromatin was diluted 10 times in a buffer containing 1% Triton, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl (pH 8.1), and incubated overnight with antibodies. Sepharose beads (Amersham Pharmacia Biosciences) were then added to the samples together with yeast tRNA as non-specific competitors. After 4 h, beads were serially washed using 1mL of washing buffer I (2mM EDTA, 20mM Tris-HCl (pH8.1), 0.1% SDS, 1% Triton X-100, 150mM NaCl), washing buffer II (2mM EDTA, 20mM Tris-HCl (pH 8.1), 0.1% SDS, 1% Triton X-100, 500 mM NaCl), washing buffer III (1mM EDTA, 10mM Tris-HCl (pH8.1), 1% NP-40, 1% deoxycholate, 0.25M LiCl) and then twice with 1mM EDTA, 10mM Tris-HCl (pH8.1), and immune complexes were eluted using 100 mL of 1% SDS and 0.1 M NaHCO3. Samples were incubated overnight at 65C to reverse cross-linking. DNA was finally purified using the NucleoSpin Extract II kit (Macherey-Nagel). Each ChIP was performed 10 times.
The (h)MeDIP, ChIP, FAIRE and input libraries were prepared using the TruSeq ChIP Sample Prep Kit (Illumina, ref. IP-202-1012). Libraries were sequenced by Illumina HiSeq following the manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Reads were mapped to the reference genome (mouse mm8) by Bowtie (Langmead et al. Genome Biol. 2009) using parameters -l 28 -n 1 -m 1 --best --strata.
The .sam files were converted to .bam files using SAMtools and the bam files were then processed to generate .wig files using MACS 1.4.0. Note that the provided .wig files are not normalized.
Genome_build: mm8
Supplementary_files_format_and_content: wig files
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| Submission date |
Jun 06, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gilles Salbert |
| Organization name |
University Rennes 1
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| Department |
IGDR UMR6290
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| Street address |
263 Av. Gal. Leclerc
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| City |
Rennes |
| ZIP/Postal code |
35200 |
| Country |
France |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE82314 |
Cytosine modifications modulate the chromatin architecture of transcriptional enhancers |
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| Relations |
| BioSample |
SAMN05210761 |
| SRA |
SRX1824743 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2188917_P19_input_RA_24h.wig.gz |
449.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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