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| Status |
Public on May 17, 2010 |
| Title |
ESC_H3K27me3_2 |
| Sample type |
SRA |
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| Source name |
R1 embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
chip antibody: H3K27me3
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| Growth protocol |
Mouse ES cell lines, R1 (passages 12-15) and E14TG2a (passages 19-21), were cultured on gelatin-coated surfaces in standard ES cell media (DMEM supplemented with 15% FBS, 1mM sodium pyruvate, 50U/ml penicillin, 50mg/ml streptomycin, 50mM 2-mercaptoethanol, 0.1mM non-essential amino acids (NEAA), 2mM glutamax (all from Invitrogen) and 1000 U/ml LIF. TS cell lines, A4 (passages 6-15) and G3 (passages 7-14), were derived from E3.5 blastocysts obtained from ICR x B5/EGFPtg/tg matings as previously described (Tanaka). TS cells were maintained on gelatin-coated surfaces in RPMI (Sigma) supplemented with 20% FBS, 1mM sodium pyruvate, 50U/ml penicillin, 50mg/ml streptomycin, 50mM 2-mercaptoethanol, 2mM glutamax (all from Invitrogen), 25ng/ml FGF4 (R&D Systems) and 1mg/ml heparin (Sigma), with 70% of the media pre-conditioned by mitotically-inactivated embryonic fibroblasts. To induce TS cell differentiation, 1 x 105 cells / cm2 were plated in standard TS cell conditions for 24h, rinsed twice with PBS and cultured for six days in unconditioned TS cell media without FGF4 and heparin. Media was changed every two days. XEN cell lines, F3 (passages 7-12) and F4 (passages 7-14), were derived from E3.5 blastocysts obtained from ICR x B5/EGFPtg/tg matings as previously described (Kunath). XEN cells were maintained on gelatin-coated surfaces in TS cell media (70% pre-conditioned) without FGF4 or heparin. Protocols can be found at http://www.sickkids.ca/research/rossant/custom/stemCells.asp
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native (unfixed) ChIP to analyze histone modifications was performed as described (Umlauf et al., 2004). Briefly, 10-50 million cells were lysed in 0.2% (v/v) IGEPAL CA-630 (Sigma) for 10min on ice, nuclei were collected by centrifugation at 10000 x g for 20min at 4°C and suspended in digestion buffer at 1mg/ml. Chromatin aliquots (0.5mg in 500ul) were digested with 10U micrococcal nuclease (GE Healthcare) for 6-9min at 37°C and soluble chromatin recovered by overnight dialysis followed by centrifugation. Successful chromatin fractionation (sample contains predominantly mononucleosomes to tetranucleosomes) was verified by agarose gel electrophoresis. Fragmented chromatin (35ug per ChIP) was immunoprecipitated overnight at 4°C with 2-10ug of one of the following antibodies: H3K4me3 (Abcam, ab8580), H3K9me2 (Millipore, 07-212), H3K9me3 (Abcam, ab8898), H3K27me3 (Millipore, 07-449), 1ul H3K27me3 (Peters et al., 2003), H3K79me3 (Abcam, ab2621), H4K20me3 (Millipore, 07-463), H3K9 acetylation (Millipore, 07-352) or rabbit anti-mouse IgG (Jackson, 315-005-003). Protein A-Sepharose (100ul of 50% (v/v) slurry; GE Healthcare) was added to each sample and rotated for 4h at 4°C. Unbound material was removed, Sepharose beads washed in salt buffer (increasing sodium chloride concentration from 75-175mM) and chromatin eluted from the beads with two 15min incubations in 1% SDS at room temperature. DNA from bound, unbound and input samples was extracted by two rounds of phenol/chloroform and precipitated with isopropanol overnight at -20°C with 30ug glycogen (Roche) as carrier. Air-dried DNA pellets were reconstituted in TE buffer. 5 Throughout the procedure, all solutions were ice-cold and freshly supplemented with protease inhibitors (Mini Complete-EDTA free, Roche) and 5mM sodium butyrate.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Chromatin IP against H3K27me3
To prepare samples for Illumima sequencing, the concentration of immunoprecipitated DNA was determined using Quant-iT PicoGreen dsDNA reagent (Invitrogen) and 100ng of each sample was sent to BC Cancer Agency Genome Sciences Centre, Vancouver, Canada, where the service was performed.
The wig processed data files are thresholded (FDR 0.0001) and unthresholded.
thresholded: 208AJAAXX_5_NCBI-Build-36_ht10.wig.gz
unthresholded: 208AJAAXX_5_NCBI-Build-36_ht2.wig.gz
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| Data processing |
Reads were aligned to NCBI build 36 by standard protocols at UBC sequencing center.
Raw ChIP-sequencing data was processed using FindPeaks (version 2.1.4 (Fejes et al., 2008)) with a false discovery rate of 0.001, an average fragment size of 174bp and a minimum peak height of two. A Java program was written to read the output Wig files and generate a BED file that contained information on the maximum peak height of all thresholded K4me3 or K27me3 regions. Significant ChIP-sequencing peaks within +/- 1kb of the TSS for K4me3 and +/- 5kb of the TSS for K27me3 were included in the final dataset. All these data were imported as tables in to a MySQL database.processed using FindPeaks (version 2.1.4; Fejes 2008 Bioinformatics) with a false discovery rate of 0.001.
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| Submission date |
Apr 14, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Joseph Cox |
| E-mail(s) |
[email protected]
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| Organization name |
University of Toronto
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| Department |
Physiology
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| Lab |
Cox System Biology
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| Street address |
1 King's College Circle, Rm 3360
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S1A8 |
| Country |
Canada |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE15519 |
Expression and ChIP-seq analyses of embryonic stem cells, extraembryonic endoderm stem cells, and trophoblast stem cells |
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| Relations |
| SRA |
SRX018520 |
| BioSample |
SAMN00010691 |
| Named Annotation |
GSM392047_208AJAAXX_5_NCBI-Build-36_ht10.wig.gz |
| Named Annotation |
GSM392047_208AJAAXX_5_NCBI_Build_36_ht2.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM392047_208AJAAXX_5_NCBI-Build-36_ht10.wig.gz |
777.0 Kb |
(ftp)(http) |
WIG |
| GSM392047_208AJAAXX_5_NCBI_Build_36_ht2.wig.gz |
19.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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