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| Status |
Public on Jun 24, 2011 |
| Title |
Integrating 5 Hydroxymethylcytosine into the Epigenetic Landscape of Human Embryonic Stem Cells 5hmC |
| Sample type |
SRA |
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| Source name |
H1 human ES cell
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| Organism |
Homo sapiens |
| Characteristics |
treatment: 5-hmC enriched genomic DNA
cell line: H1
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| Growth protocol |
H1 human ES cells were maintained on mitomycin C-treated STO cells in ES medium consisting of DMEM/F12 medium (Invitrogen) supplemented with 20% serum replacement (SR; Invitrogen), 1 mM L-glutamine (Invitrogen), 100 μM nonessential amino acids (Invitrogen), 0.1 mM Ã-mercaptoethanol (Sigma), 1X Antibiotics-Antimycotic (Invitrogen), and 4 ng/mL bFGF (Invitrogen).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified from tissue using Wizard genomic DNA purification kit (Promega) with additional Proteinase K treatment and rehydrated in 10 mM Tris (pH 7.9). Genomic DNA samples were sonicated to 100-500 bp by Misonix sonicator 3000 (using microtip, 3 pulses of 30 seconds each with 2 min of rest and a power output level of two). 5-hmC labelling reactions were performed in a 100-µL solution containing 50 mM HEPES buffer (pH 7.9), 25 mM MgCl2, 300 ng/μL sonicated genomic DNA (100-500 bp), 250 μM UDP-6-N3-Glu or UDP-Glu, and 2.25 μM wild-type BGT. The reactions were incubated for 1 hr at 37 °C. After the reaction, the DNA substrates were purified by Qiagen DNA purification kit or by phenol-chloroform precipitation and reconstituted in H2O. Biotin addition by click chemistry was performed by adding 150 μM dibenzocyclooctyne modified biotin into the DNA solution, and the reaction mixture was incubated for 2 hr at 37 °C. The DNA samples were then purified by Qiagen DNA purification kit. Biotin Labeled 5-hmC DNAs were purified/enriched by Pierce Monomeric Avidin Kit (Thermo) following manufacturerâs recommendations. After elution, the biotin-5-N3-gmC containing DNA was concentrated by 10 K Amicon Ultra-0.5 mL Centrifugal Filters (Millipore) and purified by Qiagen DNA purification kit. 25 ng genomic DNA, 5-hmC-captured DNA, or control captured DNA (in the absence of biotin) were used to initiate library preparation following the Illumina protocol for âPreparing Samples for ChIP Sequencing of DNAâ (Part# 111257047 Rev. A). DNA fragments ~150-300 bp were gel purified after the adapter ligation step and amplified by 18 cycles PCR.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
5hmC Capture and Sequencing
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| Data processing |
Processed data not provided. It will be included in the publication.
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| Submission date |
Jun 23, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Keith E Szulwach |
| E-mail(s) |
[email protected]
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| Phone |
404-712-0796
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| Organization name |
Emory University
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| Department |
Human Genetics
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| Lab |
Jin Lab
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| Street address |
615 Michael Street, Whitehead Suite 301
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE30173 |
Integrating 5 Hydroxymethylcytosine into the Epigenetic Landscape of Human Embryonic Stem Cells |
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| Relations |
| SRA |
SRX080213 |
| BioSample |
SAMN00631252 |
| 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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