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| Status |
Public on Mar 07, 2017 |
| Title |
mESC_H3K4me3 |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cell
chip antibody: H3K4me3
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| Growth protocol |
mouse ES cells were cultured with Mitomycin C (Sigma-Aldrich, St. Louis, MO)-treated STO feeder cell layer on 0.1% gelatin (Sigma-Aldrich, St. Louis, MO)-coated tissue culture dish. Fibroblast cells were cultured with Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were resuspended with digestion buffer (50mM Tris-HCL, pH7.6, 1mM CaCl2, 0.2% triton X-100) and 5mM butyrate, 0.5mM PMSF, x proteinase inhibitor. The chromatins were digested with Mnase and enzyme was inactivated by adding the stop buffer (10mM Tris, pH7.6, 5mM EDTA). The chromatin was immunoprecipitated with antibodies specific to the H3K4me3 (ab8580, Abcam, Cambridge, UK), and H3K27me3 (07-449, Millipore, Billerica, MA) in RIPA buffer (10 mM Tris, pH7.4, 1mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, and 1% Triton X-100). Bead complex was captured by magnetic stand and washed sequentially twice with RIPA buffer, RIPA + 0.3 M NaCl, and LiCl buffer (0.25 M LiCl, 0.5% NP40, 0.5% Na- Deoxycholate) and once with 1X TE + 0.2% Triton X-100, and 1X TE. Proteinase K digestion and phenol/chloroform extraction was performed to isolate the DNA.
Sequencing libraries were manually generated for high-throughput sequencing. Either end of DNA strands was filled using the Epicentre DNA END-Repair kit (Epicentre Biotechnologies, Madison, WI). Adenine was added at 3’ ends by Taq DNA polymerase (New England Biolabs, Beverly, MA) under dATP conditions. Illumina’s adaptor oligomers was ligated at both ends using Quick Ligation kit (Qiagen, Germantown, MD). After purified twice, DNA libraries are amplified using Solexa primers (Illumina, San Diego, CA) and Phusion PCR master mix (Thermo Scientific, Hudson, NH).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Illumina Casava1.6 software used for basecalling.
Sequenced reads were mapped to mm9 genome using CASAVA 1.6
The following reads were removed for down-stream analysis: multiple match, mismatch >2
Sequencing tags were shifted by +75 bp for positive strand and -75 bp for negative strand. The shifted sequencing tags were counted in non-overlapping 200bp genomic windows.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files with BED graph format
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| Submission date |
Jul 01, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Tae-Young Roh |
| Organization name |
Ewha Womans University
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| Department |
Life Sciences
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| Lab |
Sysgem Genomics
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| Street address |
52 Ewhayeodae-gil, Seodaemun-gu
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| City |
Seoul |
| State/province |
--- Select One --- |
| ZIP/Postal code |
03760 |
| Country |
South Korea |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE58965 |
Identification of early and late responder genes during cell reprogramming of mouse fibroblasts into induced pluripotent stem cells |
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| Relations |
| BioSample |
SAMN02898929 |
| SRA |
SRX642742 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1423148_mESC_H3K4me3.bedgraph.gz |
4.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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