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| Status |
Public on Mar 21, 2013 |
| Title |
CRL2097duplicate |
| Sample type |
SRA |
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| Source name |
fibroblasts
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| Organism |
Homo sapiens |
| Characteristics |
cell line: CRL2097
cell type: fibroblasts
modification: 5hmC-chemical label pull down
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| Growth protocol |
The fibroblasts were cultured in DMEM medium containing 10% FBS, 1× Non-Essential amino acids, 1× glutamine, and 1× Pen/Strep. The stem cells were maintained in hESC/hiPSC standard medium (DMEM/F12, 20% KnockOut Serum Replacement, 1× MEM Non-Essential Amino Acids, 1× glutamine, 0.11 mM 2-mercaptoethanol, 10 ng/ml bFGF) on irradiated MEF feeders.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted and purified with the DNeasy Kit (Qiagen). Genomic DNA (20ug-30ug) from each cell line was sonicated to an average size of 200bp by the Covaris sonicator. 5hmC labeling reactions were performed according to the previous protocol (Song et al., 2011) with some modifications. Briefly, the UDPG-N3 transfer was carried out with 1X reaction buffer containing 50 mM HEPES (pH 7.9) and 25 mM MgCl2, 100 uM of UDP-6-N3-Glu, and 2 uM of wild-type beta-glucose transferase for 1 h at 37° C. The labeled DNA was purified by the QIAquick PCR purification kit (Qiagen). The click chemistry was performed with the addition of 150 uM of disulfide-biotin linker, and the mixture was incubated for 2 h at 37° C. The DNA samples were then purified by the Pierce Monomeric Avidin Kit (Thermo) following the manufacturer’s recommendations. Subsequently, the 5hmC enriched DNA was concentrated by 10 K Amicon Ultra-0.5 mL Centrifugal Filters (Millipore), then purified and eluted with 12ul H2O by the MinElute PCR Purification Kit (Qiagen).
Approximately 50ng (5ul) of each 5hmC enriched DNA was used for Illumina SR library preparation by NEBNext ChIP-Seq Sample Prep Reagent Set 1(NEB) according to the manufacturer's standard protocol.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
5hmC-capture-seq
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| Data processing |
Bowtie aligned
remove duplicate reads
MACS peak calling
Genome_build: hg18/NCBI36
Supplementary_files_format_and_content: bed files with 5hmC enrichment peaks
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| Submission date |
Apr 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Tao Wang |
| E-mail(s) |
[email protected]
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| Phone |
4047270405
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| Organization name |
Emory University
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| Department |
Human Genetics
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| Lab |
Warren Lab
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| Street address |
615 Michael Street
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE37050 |
5-hydroxymethylcytosine-mediated epigenetic modifications between iPSCs and hESCs |
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| Relations |
| SRA |
SRX135684 |
| BioSample |
SAMN00849573 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM909335_CRL2097duplicate.fastq._peaks.bed.gz |
1.8 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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