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| Status |
Public on Oct 21, 2014 |
| Title |
Oocyte-rep2 |
| Sample type |
SRA |
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| Source name |
Oocyte
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| Organism |
Mus musculus |
| Characteristics |
genotype: WT
parent-of-origin: maternal
treatment: mock
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| Growth protocol |
When collecting pronuclei of zygotes, 7-10 week-old CKO (Zp3-Cre, f/-) or WT (f/f) females were superovulated. MII oocytes were collected from the mice, transferred into HTF medium supplemented with 10 mg/ml bovine serum albumin (BSA; Sigma-Aldrich), and inseminated with activated spermatozoa obtained from the caudal epididymides of adult C57BL/6J male mice. Spermatozoa capacitation was attained by 1 h incubation in HTF medium. Zygotes were cultured in a humidified atmosphere of 5% CO2/95% air at 37.8°C. Five hours after fertilization, zygotes were cultured in KSOM (Millipore). Twelve hours post-fertilization (hpf), zygotes were transferred into M2 media containinig 5 µM cytochalasin B. Zona pellucidae were cut by a Piezo impact-driven micromanipulator (Prime Tech Ltd., Ibaraki, Japan) and pronuclei of the zygotes were isolated. The parental pronuclei were distinguished by (1) the distance from the second polar body and (2) the pronuclear size. The isolated paternal and maternal pronuclei were frozen at 13 hpf in -80°C. For aphidicolin treatment, zygotes were transferred into KSOM containing 3 µg/ml aphidicolin (Sigma-Aldrich) at 5 hpf.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5 µl of lysis buffer (20 mM Tris-EDTA [pH 8.0], 20 mM KCl, and 0.3% Triton X-100, 1 mg/ml QIAGEN Protease) was directly added to the frozen samples (in < 1µl PBS) followed by 3 hours of incubation at 50 ℃ and 30 minutes of inactivation at 75 ℃. Then to the same tube, 13 µl of MspI digestion master mix containing 10 units of MspI (Thermo Scientific) and 1.8 µl 10x Buffer Tango (Thermo Scientific) was added followed by 3 hours of incubation at 37 ℃ and 20 minutes of inactivation at 80 ℃.
The digested DNA was then filled-in and tailed with an extra A to the 3’ end by adding 2 µl of end-preparation master mix containing 5 units of Klenow Fragment (exo-; Thermo Scientific), 1.8 µl of 10x Buffer Tango, 0.4 mM dATP, and 0.04 mM each of dGTP and dCTP (Thermo Scientific), followed by 40 minutes of incubation at 37 ℃ and 15 minutes of inactivation at 75 ℃. Adaptor ligation was then performed by adding 5 µl of ligation master mix, which contains 30 Weiss Units of T4 DNA Ligase (HC, Thermo Scientific), 0.5 µl of 10x Buffer Tango, 5mM ATP (Thermo Scientific), and 150 nM of methylated custom adaptor (Forward: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’, Reverse: 5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’, where * indicates phosphorothioate bond). After three incubation steps (16 ℃ for 30 min, 4 ℃ overnight, and 65 ℃ for 20 min), the reaction mix was subjected to EpiTect Fast Bisulfite Conversion Kit (QIAGEN) following manufacturer’s low-concentration protocol. The bisulfite converted DNA was immediately subjected to PCR amplification using KAPA HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems) and NEBNext Primers for Illumina (New England Biolabs). Typically, 21 PCR cycles were required when starting with ~20 pronuclei. After amplification, final libraries were obtained by size selection of 150-500 bp DNA fragments using SPRIselect beads (Beckman Coulter).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
RRBS sequencing reads were mapped to the mouse genome (mm9) using Bismark v0.10.1 (Babraham Bioinformatics) after adapter trimming by Trim Galore (Babraham Bioinformatics). The methylation level of each covered cytosine in CpG context was calculated as the number of reported C divided by the total number of reported C and T.
Genome_build: mm9
Supplementary_files_format_and_content: The processed data files are the coverage files generated by Bismark (chr, start, end, methylation%, number of reported C, number of reported T)
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| Submission date |
Sep 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Li Shen |
| Organization name |
HHMI/Boston Children's Hospital/Harvard Medical School
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| Street address |
200 Longwood Ave
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| City |
Boston |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE61330 |
Role of Tet3 and DNA replication in zygotic demethylation of both paternal and maternal genomes [RRBS] |
| GSE61331 |
Role of Tet3 and DNA replication in zygotic demethylation of both paternal and maternal genomes |
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| Relations |
| Reanalyzed by |
GSE77019 |
| BioSample |
SAMN03031729 |
| SRA |
SRX699476 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1502487_Oocyte-rep2.bismark.cov.txt.gz |
9.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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