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| Status |
Public on Dec 31, 2010 |
| Title |
KSO_H3K9me3_ChIP_Seq |
| Sample type |
SRA |
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| Source name |
Chromatin IP against H3K9me3
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic fibroblasts (MEF)
transfection: Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO)
passages: p3-5
antibody: anti-H3K9me3
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| Growth protocol |
Mouse Embryonic Fibroblasts were derived from day e13 embryos. MEF and KSO were cultured in Dulbecco’s modified Eagle medium supplemented with 10% cosmic calf serum, β-mercaptoethanol, nonessential amino acids, L glutamine and penicillin/streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was done following the Agilent Mammalian ChIP-on-chip protocol (version 9.1, Nov 2006). In summary, MEF and mES cells were grown as described above and cross-linked for 10 minutes at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution (11% formaldehyde, 50mM Hepes pH7.3, 100mM NaCl, 1mM EDTA pH8.0, 0.5mM EGTA pH8.0) to the growth media followed by two washes with PBS. Cells were scraped and frozen in liquid nitrogen. 100ul of Dynal magnetic beads (Sigma) were blocked with 0.5% BSA (w/v) in PBS. Magnetic beads were bound with 10ug of the indicated antibody. Antibodies used are as follows: H3K4me3 (AbCam, Ab8580), H3K9me3 (AbCam, Ab8898) and H3K27me3 (Ab6002). Cells were resuspended, lysed in lysis buffers and sonicated to solubilize and shear crosslinked DNA. Sonication conditions vary depending on cells, culture conditions, crosslinking and equipment. For H3K9me3, the sonication buffer was Tris-HCl pH8 20mM, 150mM NaCl, 2mM EDTA, 0.1% SDS, Triton X-100 1%. We used a Misonix Sonicator 3000 and sonicated at approximately 24 watts for 10 x 30 second pulses (60 second pause between pulses). Samples were kept on ice at all times. The resulting whole cell extract was incubated overnight at 4°C with 100 μl of Dynal Protein G magnetic beads that had been pre-incubated with approximately 10 μg of the appropriate antibody. Beads were washed 4 times with: 1 time with the sonication buffer, 1 time with 20mM Tris-HCl pH8, 500nM NaCl, 2mM EDTA, 0.1% SDS, 1%Triton X-100, 1 time with 10mM Tris-HCl pH8, 250nM LiCl, 2mM EDTA, 1% NP40 and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C for 1 hour with occasional vortexing and crosslinking was reversed by overnight incubation at 65°C. Whole cell extract DNA reserved from the sonication step was also treated for crosslink reversal. RNA and protein were digested using RNAse A and Proteinase K and DNA was purified with phenol chloroform extraction and ethanol precipitation. DNA was prepared for sequencing according to a modified version of the Illumina/Solexa Genomic DNA protocol. Fragmented DNA was prepared for ligation of Solexa linkers by repairing the ends and adding a single adenine nucleotide overhang to allow for directional ligation. A 1:100 dilution of the Adaptor Oligo Mix (Illumina) was used in the ligation step. A subsequent PCR step with limited (18) amplification cycles added additional linker sequence to the fragments to prepare them for annealing to the Genome Analyzer flow-cell. After amplification, a narrow range of fragment sizes was selected by separation on a 2% agarose gel and excision of a band between 150-300 bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence). The DNA was purified from the agarose and diluted to 10 nM for loading on the flow cell. The DNA library (2-4 pM) was applied to the flow-cell (8 samples per flow-cell) using the Cluster Station device from Illumina. The concentration of library applied to the flow-cell was calibrated such that polonies generated in the bridge amplification step originate from single strands of DNA. Multiple rounds of amplification reagents were flowed across the cell in the bridge amplification step to generate polonies of approximately 1,000 strands in 1μm diameter spots. Double stranded polonies were visually checked for density and morphology by staining with a 1:5000 dilution of SYBR Green I (Invitrogen) and visualizing with a microscope under fluorescent illumination. Validated flow-cells were stored at 4 degrees C until sequencing. Flow-cells were removed from storage and subjected to linearization and annealing of sequencing primer on the Cluster Station. Primed flow-cells were loaded into the Illumina Genome Analyzer 1G. After the first base was incorporated in the Sequencing-by-Synthesis reaction the process was paused for a key quality control checkpoint. A small section of each lane was imaged and the average intensity value for all four bases was compared to minimum thresholds. Flow-cells with low first base intensities were re-primed and if signal was not recovered the flow-cell was aborted. Flow-cells with signal intensities meeting the minimum thresholds were resumed and sequenced for 26 or 32 cycles.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Images acquired from the Illumina/Solexa sequencer were processed through the bundled Solexa image extraction pipeline which identified polony positions, performed base-calling and generated QC statistics. Sequences were aligned using ELAND software to NCBI Build 36 (UCSC mm8) of the mouse genome. Only sequences that mapped uniquely to the genome with zero or one mismatch were used for further analysis. When multiple reads mapped to the same genomic position, a maximum of two reads mapping to the same position were used. Analysis methods were derived from previously published methods (PMID:19884255). Sequence reads from multiple flow cells for each IP target were combined. Each read was extended 100bp, towards the interior of the sequenced fragment, based on the strand of the alignment. Across the genome, in 25 bp bins, the number of ChIP-Seq reads within a 1kb window surrounding each bin (+/- 500bp) was tabulated. The 25bp genomic bins that contained statistically significant ChIP-Seq enrichment were identified by comparison to a Poissonian background model. Assuming background reads are spread randomly throughout the genome, the probability of observing a given number of reads in a 1kb window can be modeled as a Poisson process in which the expectation can be estimated as the number of mapped reads multiplied by the number of bins (40) into which each read maps, divided by the total number of bins available (we estimated 70%). Enriched bins within 1kb of one another were combined into regions. The Poissonian background model assumes a random distribution of background reads, however we have observed significant deviations from this expectation. Some of these non-random events can be detected as sites of apparent enrichment in negative control DNA samples and can create many false positives in ChIP-Seq experiments. To remove these regions, we compared genomic bins and regions that meet the statistical threshold for enrichment to a set of reads obtained from Solexa sequencing of DNA from whole cell extract (WCE) in matched cell samples. We required that enriched bins and enriched regions have five-fold greater ChIP-Seq density in the specific IP sample, compared with the control sample, normalized to the total number of reads in each dataset. This served to filter out genomic regions that are biased to having a greater than expected background density of ChIP-Seq reads
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| Submission date |
Apr 08, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Steve Bilodeau |
| E-mail(s) |
bilodeau@wi.mit.edu
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| Organization name |
Whitehead Institute
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| Street address |
9 Cambridge Center
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE21271 |
Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO) |
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| Relations |
| SRA |
SRX018934 |
| BioSample |
SAMN00011037 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM531816_H3K9me3_KSO_min1.5.WIG.gz |
6.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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