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| Status |
Public on Sep 15, 2016 |
| Title |
P19_H3K27me3_6h_ATRA |
| Sample type |
SRA |
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| Source name |
P19 embryonal carcinoma cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: P19
agent: ATRA
time point: 6 hours
chip antibody: Millipore 07-449
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| Treatment protocol |
For cell differentiation induction, ATRA was added to plates in a final concentration of 1x10-6 M at different time points. For RAR subtype-specific agonists’ treatment, cells were incubated with BMS961 (RARg specific; final concentration 1x10-7 M), BMS753 (RARa specific; final concentration 1x10-6 M) or BMS641 (RARb specific; final concentration 1x10-7 M).
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| Growth protocol |
F9 EC cells were cultures in Dubelcco’s modified Eagle’s medium supplemented with 10% fetal Calf serum. In a similar manner, P19 cell’s medium was supplemented with 5% fetal Calf serum and 5% delipidated Calf serum. In both cases the medium was complemented with 40ug/ml Gentamicin. F9 or P19 EC cells were cultured in monolayer conditions seeded in gelatine-coated tissue culture plates (0.1%).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
F9 or P19 EC cells were fixed with 1% para-formaldehyde (Electron Microscopy Sciences) for 30 minutes at room temperature. ChIP assays were performed following previously described conditions: chromatin sonication and immunoprecipitation in lysis buffer (50mM Tris–Cl pH=8, 140mM NaCl, 1mM EDTA, 1% Triton, 0.1% Na-deoxycholate) complemented with protease inhibitor cocktail (Roche 11873580001); 2x washes with lysis buffer; 2x washes with lysis buffer containing 360mM NaCl; 2x washes with washing buffer (10mM Tris–Cl pH=8, 250mM LiCl, 0.5% NP-40, 1mM EDTA, 0.5% Na-deoxycholate); 2x washes with 1x TE; elution at 651C; 15 min in elution buffer (50mM Tris–Cl pH=8, 10mM EDTA, 1% SDS). RXRa has been immunoprecipitated with purified polyclonal antibodies generated by immunization of rabbits with the following peptide:mRXRa: PB105 (MDTKHFLPLDFSTQVNSSSLNSPTGRGC). RXRa ChIP assays were performed with 6x106 cells per time point; while histone modification marks were evaluated with 2x106 cells. FAIRE assays were performed as described previously (Giresi et al, 2007; Simon et al, 2012).
qPCR-qualified ChIP assays were quantified (Qubit dsDNA HS assay kit; Invitrogen); then 10 ng of the ChIPed material was used for preparing Illumina sequencing libraries following a multiplexing approach (NEXTflexTM ChIP-seq Bioo Scientific; ref: 514120). Prepared sequencing libraries were sequenced on the Illumina instrument HiSeq2000 (4 ChIP samples per lane). Regular Illumina pipelines were used for image processing and base calling. Sequence files were then aligned to the mouse genome assembly following by default parameters (mm9; Bowtie).
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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 |
ChIP-seq and related datasets (FAIRE-seq; Input control) were quality certified with NGS-QC Generator tool (www.ngs-qc.org). Specifically, this methodology allows providing enrichment quality descriptors discretized in a scale ranging from “AAA” (Best) to “DDD” (worst).
Relevant binding sites in all ChIP-Seq and FAIRE-Seq datasets were identified with MeDiChISeq, a regression-based approach which after a learning process defines a representative binding pattern from the dataset under analysis (Mendoza-Parra et al; BMC Genomics 2013). Specifically a P-value confidence cutoff of 1x10-2.5 complemented by a peak intensity threshold based on a Poisson distribution background model.
Genome-wide quantile normalization prior multiprofiles comparison has been performed with EPImetheus (Saleem MA; unpublished).
Genome_build: mm9
Supplementary_files_format_and_content: peaks (MeDiChISeq) and quality report (pdf)
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| Submission date |
May 05, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Marco Antonio Mendoza-Parra |
| E-mail(s) |
marco@igbmc.fr
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| Organization name |
IGBMC
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| Street address |
1, rue Laurent Fries
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| City |
Illkirch; Strasbourg |
| ZIP/Postal code |
67400 |
| Country |
France |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE68291 |
Reconstructing divergent retinoid-induced cell fate-regulatory programs in stem cells |
| GSE68540 |
Reconstructing divergent retinoid-induced cell fate-regulatory programs in stem cells [ChIP-Seq] |
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| Relations |
| BioSample |
SAMN03603514 |
| SRA |
SRX1017439 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1674829_P19_H3K27me3_6hATRA.bed.gz |
450.8 Mb |
(ftp)(http) |
BED |
| GSM1674829_P19_H3K27me3_6hATRA.pdf.gz |
361.0 Kb |
(ftp)(http) |
PDF |
| GSM1674829_P19_H3K27me3_6h_ATRA_peaksMed.txt.gz |
575.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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