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| Status |
Public on May 20, 2013 |
| Title |
32-cell.TABseq |
| Sample type |
SRA |
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| Source name |
32-cell
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| Organism |
Danio rerio |
| Characteristics |
cell type: 32-cell
developmental stage: embryo
stain: Tubingen
library strategy: TAB-Seq
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
TAB-seq:5-hmC library was constructed as described (Yu et al., 2012). Briefly,fragmented genomic DNA with spike-in controls was ligated to methylated adapter , and was firstly glucoslyated and oxidized before bisulfite treatment. Efficient conversion of unmodified cytosine to uracil and efficient conversion of 5mC to 5caU/U were calculated by spiked M. SsI treated lambda DNA. 5hmc protection rate was calculated by spiked pUC19 DNA .
Sperms were released by gently but repeatedly disrupting the testis with a pipette tip in HBSS. Unfertilized oocytes were collected by squeezing the abdomen of females about 5 minutes after spawning. Embryos were grown in embryo medium at 28°C, staged according to standard morphological criteria..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 mMNaCl, 5 mM MgCl2, 10 µg/ml cytochalasin B, 1 mMdithiothreitol 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.
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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: TAB-Seq
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality
Zv9 reference genome was downloaded from UCSC. The 48,502 bp Lambda genome was also included in the reference sequence as an extra chromosome for calculating bisulfite conversion rate. Filtered paired-end methylC-seq reads were mapped against the reference by Bismark_v0.6.4 (Krueger and Andrews, 2011) with stringent parameters: -n 2 -l 60 -e 100 -X 600. A custom script was used to examine whether pair-end reads were overlapped and the overlapped part is trimmed from one end to prevent counting twice from the same observation.
For CpG i, define mi as the number of reads showing methylation over position i (both strands). Define ui as the number of reads showing lack of methylation over CpG i. The methylation level is estimated as mi/( mi + ui), which is an estimate of the probability that CpG i is methylated in a molecule sampled randomly from the cell population. Because CpG methylation is symmetric, mi and ui include observations associated with the cytosines on both strands for the i-thCpG.
for TAB-seq , 5-hmC mapping was performed as described (Yu et al., 2012). Paired-reads were mapped uniquely to the reference genome (Zv9, USCS) plus Lambda and pUC19 DNA sequence as extra chromosome by Bismark. All the CpG sites disrupted by SNPs were filtered from the analysis. 5hmC was called with a binomial distribution follows the method previously reported (Yu et al., 2012). A binomial distribution (Lister et al., 2009)was applied to model this probabilistic event with N as the depth of sequencing at the cytosine and p as the 5mC non-conversion rate. Efficient conversion of unmodified cytosine to uracil and efficient conversion of 5mC to 5caU/U were calculated by spiked M. SsI treated lambda DNA.
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: wig files contain methylaion level of each CpG site
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| Submission date |
May 03, 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 |
| BioSample |
SAMN02117052 |
| SRA |
SRX273748 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1133400_chrall.32-cell.TAB-seq.wig.gz |
78.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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