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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 04, 2012 |
| Title |
H3K27ac ChIP: KAP1 WT IP |
| Sample type |
SRA |
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| Source name |
H3K27ac ChIP: KAP1 WT IP
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Embryonic stem cells (ES3)
time-point: 4 days post puromycin selection of empty shRNA vector
chip antibody: H3K27ac (Abcam: ab4729)
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| Growth protocol |
ES cells were cultured in Glasgow Minimum Essential Medium (GMEM, Sigma: G5154) with sodium pyruvate (used at 1mM, Sigma: S8636), MEM non-essential amino acids (used at 1x, Gibco: 11140-035), L-glutamine (used at 2mM, Gibco: 25030-024), 2-mercaptoethanol (used at 0.1mM, Sigma), ES cell tested FBS (Gibco: 16141-079) and leukaemia inhibitory factor (LIF, used at 1,000 units/ml, Chemicon: ESG1107). Cells were grown on 0.2% gelatin (Sigma: 48723-500G-F)-coated plates and split every two days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ES cell samples were washed 2x (in PBS + 2% FCS), counted to normalize by cell number, cross-linked (ten minutes rotation in 1% formaldehyde), quenched with glycine (at 125mM on ice), washed 3x (PBS) and pelleted at 10e7 cells per ependorf. Pellets were lysed, resuspended in 1ml sonication buffer on ice (10mM Tris pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% NaDOC, 0.25% NLS and protease inhibitors), transferred to glass 12x24mm tubes (Covaris: 520056) and sonicated (Covaris settings: 20% duty cycle, intensity 5, 200cyles/ burst, 30 minutes). Sonication was then assessed by reverse cross-linking overnight in the presence of proteinase K and RNase, followed by DNA extraction and quantification on a Bioanalyzer (Agilent 2100 machine). Samples were also checked for the absence of single-stranded DNA by Exonuclease I treatment. Immunoprecipitations were performed in duplicates or triplicates with Dynabeads (100.03D) using 1-2x10e6 cells, 80ul pre-blocked beads and 5ug antibody (or no antibody as a control) per sample in IP buffer (167mM NaCl, 16.7mM Tris pH 8.1, 1.2mM EDTA, 0.5mM EGTA, 1.1%TritonX100 and protease inhibitors) overnight. After washing and reverse cross-linking (also overnight) and DNA extraction, results were quantified by SYBR green qPCR.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
no processed data
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| Data processing |
Raw reads were mapped to the mouse genome and transcriptome using Bowtie
Peaks were called using MACS
For histone modifications, enriched regions were defined using the ChIP-part suite
Genome_build: mm9
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| Submission date |
Nov 06, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Adamandia Kapopoulou |
| E-mail(s) |
adamandia.kapopoulou@epfl.ch
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| Phone |
0041216930940
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| Organization name |
EPFL
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| Department |
Sciences de la Vie
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| Lab |
Trono
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| Street address |
Station 15
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| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE41903 |
TRIM28 repression of retrotransposon-based enhancers is necessary to preserve transcriptional dynamics in embryonic stem cells |
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| Relations |
| SRA |
SRX202993 |
| BioSample |
SAMN01801733 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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