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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 28, 2015 |
| Title |
WT-Blastocyst-rep2 |
| Sample type |
SRA |
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| Source name |
WT-Blastocyst-rep2
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| Organism |
Mus musculus |
| Characteristics |
cell: blastocyst
strain: C57BL/6J x CAST/EiJ
genotype: WT
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| Growth protocol |
MII oocytes were collected from 7-10 week-old superovulated females. They were transferred into HTF medium supplemented with 10 mg/ml bovine serum albumin (BSA; Sigma-Aldrich) and inseminated with activated spermatozoa collected from the caudal epididymides of adult CAST/EiJ male mice. Spermatozoa capacitation was attained by 1 h incubation in HTF medium. Five hours after fertilization, zygotes were transferred into KSOM. Zygotes and blastocysts were collected at 13 and 96 hours postfertilization, respectively and frozen at -80°C until use. Biological duplicates for each samples were used and each sample contains 40-50 zygotes or 4 blastocysts.
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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). 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 |
We first extracted discernible paternal reads from the total reads using SNP information between the CAST and C57BL/6J mice. These paternal RRBS reads were then 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 from the discernible paternal reads (chr, start, end, methylation%, number of reported C, number of reported T)
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| Submission date |
Oct 26, 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 (1) |
| GSE62719 |
Haploinsufficiency, but not defective paternal 5mC oxidation, accounts for the developmental defects of maternal Tet3 knockouts |
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| Relations |
| BioSample |
SAMN03144439 |
| SRA |
SRX743636 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1532322_CAST-WT-Blastocyst-rep2.cov.txt.gz |
885.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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