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| Status |
Public on Jun 30, 2013 |
| Title |
meDIP-Seq vitamin C treated 12hr |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells, vitamin C treated
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| Organism |
Mus musculus |
| Characteristics |
cell type: Oct4-GiP ES cells
treatment: vitamin C treated
time point: 12hr
antibody target: mouse monoclonal anti-5-methylcytosine
antibody vendor: Eurogentec
antibody cat#: BI-MECY-0100
antibody lot#: 100615
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| Treatment protocol |
ES cells were cultured in the presence or absence of Vitamin C (L-ascorbic acid 2-phosphate, 100 μg/ml) for 12 or 72 hours. Vitamin C was added to ES cells one day after seeding. Medium with or without Vitamin C supplementation was replaced daily.
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| Growth protocol |
ES cells were cultured on 0.1% gelatin coated plates in N2B27 medium supplemented with the MEK inhibitor PD0325901 (1μM, Stemgent), and the GSK3β inhibitor CHIR99021 (3μM, Selleck Chemicals), and LIF (1000U/ml, Millipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Phenol-chloroform-isoamyl alcohol extraction
For each sample, 12μg of genomic DNA was isolated, split into 3 replicates of 4ug each and sonicated to ~100-500 bp on a Covaris E210 platform (75s, 10% duty cycle; Covaris Inc.;). Sheared DNA was end-repaired, A-tailed, and ligated to custom paired-end adapters as described <Morin et al., Nature 2011>. Ligated gDNA was size selected by 8% PAGE to remove unligated adapters (100-300bp), replicates pooled and subjected to qPCR using truncated PE1.0/2.0 PCR primers to assess ligation efficiency and quantified (quBit). Adapter-ligated DNA from each sample was split into three equal replicates (0.5-1.5ug) and immunoprecipitation using mouse monoclonal anti-methylcytidine antibody (1 mg/ml, Eurogentec, catalog # BI-MECY-0100), mouse monoclonal anti-hydroxymethylcytidine (hmC) antibody (Diagenode, EF-131-0050) or a nonspecific mouse IgG IP (Jackson Immunoresearch) as a negative control. Synthetic hmC containing DNA was spiked into the hmC replicates to monitor immunoprecipitation efficiency. Adapted DNA was heat denatured at 95 ° C for 10 minutes, rapidly cooled on ice, and immunoprecipitated with 1 μl primary antibody per microgram of DNA overnight at 4° C with rocking agitation in 500 μl IP buffer (10 mM sodium phosphate buffer, pH 7.0, 140 mM NaCl, 0.05% Triton X-100). To recover the immunoabsorbed DNA fragments, 1 μl of rabbit anti-mouse IgG secondary antibody (2.5 mg/ml, Jackson Immunoresearch) and 100 μl Protein A/G beads (Pierce Biotechnology) were added and incubated for an additional 2 hr at 4° C with agitation. After immunoprecipitation a total of 6 IP washes were performed with ice cold IP buffer. Washed beads were resuspended in TE with 0.25% SDS and 0.25 mg/ml proteinase K for 2 hrs at 55° C and then allowed to cool to room temperature. Immunoprecipitated and supernatant DNA were purified using Qiagen MinElute columns and eluted in 16 μl EB (Qiagen, USA). Fifteen cycles of PCR were performed on 5 μl of the immunoprecipitated DNA using the single end Illumina PCR primers. The resulting reactions are purified over Qiagen MinElute columns, after which a final size selection (192 -392 bp) was performed by electrophoresis in 2% agarose. Libraries were QC’d by spectrophotometry and Agilent DNA Bioanalyzer analysis. An aliquot of each indexed library was pooled by sample and sequenced 2 per lane on an Illumina Hiseq2000 platform (2X76nt + 7nt) following the manufacturer’s recommended protocol and V3 chemistry (Illumina Inc.).
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Base-calling software: Illumina RTA 1.12.4.2 <default>
Read Alignment Software: BWA v0.5.7 <default>
Read Sorting: Samtools
Mark Duplicates: Picard 1.60
Wiggle Track Generation: Findpeaks 4.01 <unthresholded>
Genome_build: genome build: mm9
Supplementary_files_format_and_content: Unthreasholded Wiggle Files
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| Submission date |
Apr 26, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Martin Hirst |
| E-mail(s) |
mhirst@bcgsc.ca
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| Phone |
604-8226673
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| Organization name |
BCCA Genome Sciences Centre
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| Department |
Epigenomics
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| Street address |
675 West 10th Avenue
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| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 1L3 |
| Country |
Canada |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE46402 |
Vitamin C induces Tet-dependent DNA demethylation in ES cells to promote a blastocyst-like methylome [MeDIP-Seq] |
| GSE46403 |
Vitamin C induces Tet-dependent DNA demethylation in ES cells to promote a blastocyst-like methylome |
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| Relations |
| BioSample |
SAMN02058384 |
| SRA |
SRX272064 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1129773_A17979.wig.gz |
89.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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