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| Status |
Public on May 20, 2013 |
| Title |
16cell.5hmc.hMeDIP |
| Sample type |
SRA |
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| Source name |
embryo
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| Organism |
Danio rerio |
| Characteristics |
developmental stage: 16cell
stain: Tubingen
tissue: embryo
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Zebrafish embryos were chilled on ice, washed by pre-chilled PBS buffer three times to avoid the contamination and homogenized in 10 vol buffer N (10 mM HEPES, pH 7.5, 250 mM sucrose, 50 mM NaCl, 5 mM MgCl2, 10 µg/ml cytochalasin B, 1 mM dithiothreitol and protease inhibitors) with ten strokes of a loose fitting pestle in a glass homogenizer, and lysates were decanted for 20 min on ice. Then lysates were filtered with 70µm cell strainer（BD Biosciences) and centrifuged through 1 M sucrose at 1000 g for 30 min. Pelleted nuclei from embryos were subjected to genomic DNA extraction by standard phenol precipitation method.
5-hmC contained DNA were enriched as described(Robertson et al., 2012) with modifications. Fragmented DNA was then subjected to end repair with the end repair enzyme mix (NEB), dA tailing with the Klenow 3´-5´ exo- (NEB) and ligation of multiplexing adapters (Illumina) using T4 DNA ligase (NEB). Adapter-ligated DNA of 280-500bp was purified by 2％ agarose gel electrophoresis, and then 5hmC was converted to β-glucosyl-5-hmC (β-glu-5-hmC) by T4 β-glucosyltransferase. β-glu-5-hmC contained DNA fragment was further pulled down by J-binding protein 1 (JBP1)-coated magnetic beads from Quest 5-hmC™ DNA Enrichment Kit (zymo research). Finally, enriched DNA fragments that have adapters on both ends were amplified using PCR to create the final sequencing library and sequenced by Illumina HiSeq 2000. A quantity of fragmented genomic DNA was set aside as input DNA. The efficiency of precipitation was checked by kit included positive control fragments.
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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 |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality
FASTQ sequence files were aligned to the Zebrafish reference (Zv9) using BWA Version: 0.6.1-r104, retaining only unique, non-duplicate genomic matches with no more than 2 mismatches within the first 32bp.
5-hmC peaks were identified using MACS with the following parameters: effective genome size = 1.40e+09; band width = 200; Tag size = 101; P-value cutoff = 1.00e-05; model fold= 4,20; ranges for calculating regional lambda are: peak_region, 200, 1000.
all the processed files are the raw results got from MACS without any filter step.
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: tab-delimited text files include peak location for each Sample …
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| Submission date |
Feb 05, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Lan Jiang |
| E-mail(s) |
lan_jiang@med.harvard.edu
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| Organization name |
Beijing Institute of Genomics
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| Lab |
Liu Jiang
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| Street address |
NO.1 Beichen West Road, Chaoyang District
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| City |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE44075 |
Sperm but not oocyte DNA methylome is inherited by zebrafish early embyros |
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| Relations |
| SRA |
SRX225359 |
| BioSample |
SAMN01911302 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1077595_16cell.vs16input_peaks.txt.gz |
1.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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