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| Status |
Public on Aug 08, 2013 |
| Title |
SyKOyNyD1 |
| Sample type |
SRA |
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| Source name |
Reprogramming intermediates
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| Organism |
Mus musculus |
| Characteristics |
cell type: Reprogramming intermediates
modification: 5hmC-chemical label pull down
reprogramming method: OySyNyK
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| Treatment protocol |
Genomic DNA sonicated to fragments shorter than 500 bp was labeled with azide UDPG catalyzed by beta-glucosyl transferase for 1 h at 37C. The labeled DNA fragments were purified using a PCR purification kit (Qiagen), and then eluted in nuclease-free water. The labeled DNA fragments were biotinylated with a biotin-bisulfide linker at 37C for 2 h. After purification with the PCR purification kit and elution in 100 ul nuclease-free water, the biotinylated DNA was incubated with Dynabeads MyOne Streptavidin C1 (Cat#: 650.02; Invitrogen) in binding buffer containing 10 mM Tris-HCl, pH 7.5, 1 M NaCl, 1 mM EDTA, and 0.5% Tween 20 at room temperature for 15 min. After thorough washing with binding buffer, the captured DNA was eluted with 50 mM fresh DTT at room temperature for 2 h. A Bio-spin 6 Tris column (Cat#: 732-6227; Bio-Rad) was used to remove the DTT from the elution buffer, followed by purification using a Qiagen MinElute PCR Purification Kit, and then the captured DNA was eluted in 10 ul elution buffer.
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| Growth protocol |
For OG2-MEFs, the above medium was further supplemented with 0.055 mM β-mercaptoethanol (Gibco), 2 mM L-GlutaMax (Gibco) and 0.1 mM non-essential amino acids (NEAA; Gibco), and for iPSC generation, 50 µg/ml vitamin C (Sigma) and 1000 U/ml LIF (Millipore) were added. iPSCs were maintained on mitomycin C-treated MEF feeder cells knockout DMEM supplemented with 15% knockout serum replacement, 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), 0.055 mM β-mercaptoethanol (Gibco), 2 mM L-GlutaMax (Gibco), 0.1 mM NEAA (Gibco), and 1000 U/ml leukemia inhibitory factor (Millipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The captured DNA fragments were used for generation of libraries following the Illumina protocol for “Preparing Samples for ChIP Sequencing of DNA” (Part# 111257047 Rev. A). A total of 25 ng input genomic DNA, 5hmC-captured DNA or ChIP-DNA was used to initiate the protocol.
DNA fragments (~150–300 bp) were selected using AMPure beads after the adaptor ligation step. PCR-amplified DNA libraries were quantified on an Agilent 2100 Bioanalyzer and diluted to 6–8 pM for cluster generation and sequencing
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Library strategy: 5hmC-Seq
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 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Tao Wang |
| E-mail(s) |
twang9@emory.edu
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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 |
GPL13112 |
| Series (1) |
| GSE46202 |
Coordination of engineered factors with TET1/2 promotes early stage epigenetic modification during somatic cell reprogramming |
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| Relations |
| BioSample |
SAMN02048978 |
| SRA |
SRX268484 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1126194_SyKOyNyD1.fastq._peaks.bed.gz |
1.4 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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