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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 30, 2008 |
| Title |
H3K36me3_mES_rep1b |
| Sample type |
SRA |
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| Source name |
ESC
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| Organism |
Mus musculus |
| Characteristics |
Chromatin immunoprecipitation for H3K36me3
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| Treatment protocol |
Refer to manuscript
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| Growth protocol |
V6.5 (C57BL/6-129) murine ES cells were grown under typical ES conditions on irradiated mouse embryonic fibroblasts (MEFs). For location analysis, cells were grown for one passage off of MEFs, on gelatinized tissue-culture plates. Cells harbouring a Dox-repressible Oct4 allele (ZHBT-c4 cells) were treated with 2mg/ml doxycycline (SIGMA, D-9891) for 12hrs or 24hrs. To generate neural precursor cells, ES cells were differentiated along the neural lineage using standard protocols. V6.5 ES cells were differentiated into neural progenitor cells (NPCs) through embryoid body formation for 4 days and selection in ITSFn media for 5–7 days, and maintained in FGF2 and EGF2 (R&D Systems). Mouse embryonic fibroblasts were prepared and cultured from DR-4 strain mice.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq samples: Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using different antibodies. Purified immunoprecipitated DNA were prepared for sequencing according to a modified version of the Solexa Genomic DNA protocol. Fragmented DNA was end repaired and subjected to 18 cycles of LM-PCR using oligos provided by Illumina. Amplified fragments between 150 and 300bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence) were isolated by agarose gel electrophoresis and purified.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
V6.5 murine ES cells were cultured as described in Boyer et al., Nature 2006.
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| Data processing |
Images analysis and base calling was done using solexa pipeline and reads aligned to both mouse NCBI build 36 and 37 using ELAND. For miRNA analysis - 5p end defined by start of tag, 3p end defined by 6nt perfect match to 3p linker and . Alignments to build 37 were used for analysis of the mmu-mir-290 cluster only as that cluster is not represented on build 36. For ChIP-Seq, sequences from all lanes were extended 200bp (maximum fragment length accounting for ~100bp of primer sequence), and allocated into 25 bp bins. Genomic bins containing statistically significant ChIP-seq enrichment were identified by comparison to a Poissonian background model, using a p-value threshold of 10-9. Additionally, we used an empirical background model obtained from identical Solexa sequencing of DNA from whole cell extract (WCE) from matched cell samples (>5X normalized enrichment across the entire region).
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| Submission date |
Jul 23, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
Whitehead Institute |
| E-mail(s) |
sgupta@wi.mit.edu
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| Phone |
617-324-0339
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| Organization name |
Whitehead Institute
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| Department |
Genome Technology Core
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| Street address |
9 Cambridge Center
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02141 |
| Country |
USA |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE11724 |
Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells |
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| Relations |
| SRA |
SRX003856 |
| BioSample |
SAMN02195337 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM307153_processedcounts.txt.gz |
53.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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