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| Status |
Public on Mar 23, 2017 |
| Title |
212_pronucleus_w-o-inh-male |
| Sample type |
SRA |
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| Source name |
zygote
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
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| Growth protocol |
3-4-week-old C57BL/6J females were superovulated by intraperitoneal injection of PMSG (pregnant mare's serum gonadotropin; 5 IU, Folligon, Intervet) followed by hCG (human chorionic gonadotropin; 5 IU, Chorulon, Intervet) injection 48 hours later. Natural matings to C57BL/6J males were set up overnight. Zygotes were released from the ampullae and treated with hyaluronidase (Sigma) to remove surrounding cumulus cells. For isolation of maternal and paternal nuclei, zygotes were pre-incubated in culture medium supplemented with 5 μg/ml cytochalasin B (Sigma) and 1 μM nocodazole (Sigma) for 15 minutes. The zona pellucida of the zygotes was opened and nuclei were extracted one by one. The maternal and paternal origin of the nuclei was determined taking size of the nuclei and position in relation to second polar body into account.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei were fixed in separate drops of M2 (not containing BSA) with 2% formaldehyde for 15 minutes. Nuclei were washed through drops of complete M2. For zygote experiments without pronuclear extraction, zygotes were treated identically to oocytes. Here nuclei were separated into individual wells after SDS treatment and kept separate in the subsequent steps of the protocol. After fixation, zygotes or single isolated pronuclei were incubated in 9 μl of lysis buffer for at least 15 minutes on ice. Similarly zygotes or pronuclei were incubated in 9 μl of 1× NEB3 buffer supplemented with 0.6% SDS for 2 hours at 37° with shaking in humidified atmosphere. Then nuclei were incubated in 1× DpnII buffer (NEB) with 1× BSA and 5 U DpnII (NEB). After overnight incubation in a humidified incubator at 37° the DNA ends were ligated in 9 μl of 1× T4 DNA ligase buffer containing 5 U T4 DNA ligase (Thermo Scientific) and they were incubated at 16°C for 4.5 hours with 50 rpm rotation, and then were kept for 30 min at room temperature. DNA from each nucleus was amplified using illustra GenomiPhi v2 DNA amplification kit (GE Healthcare) or illustra Single Cell GenomiPhi DNA amplification kit according to manufacturer's recommendations, but both protocols were modified to include a decrosslinking step of an overnight (14-16 hours) incubation at 65°C. After the whole-genome amplification procedure, the DNA was purified. 1 μg (or all available amount, if the yield was lower) of DNA was diluted to 500 μl with sonication buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.1% SDS) and was sonicated using Branson Sonifier 150 for 20 seconds with power setting 3. The sonicated DNA was purified and then submitted for library preparation and sequencing.
The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample preparation protocol for Solexa platforms (NEB reagents).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Base calling was performed by VBCF NGS unit
Mapping: iterative mapping (see Imakaev et al, 2012) using bowtie2 with increased stringency parameters: --score-min -L 0.6,0.2--very- sensitive
Read filtering: if two reads map to the same strands, and each side of the read is within 500 bp of any side of the other read, we retained only one copy of the read.
Additional filtering: we separated the genome into 500 bp bins. If any two bins were interacting more than once, only one interaction was counted. Since each region is only present in the single cell up to 4 times (in oocytes), we eliminated all 500 bp bins that form more than 8 unique interactions with other bins.
Filtered reads were then binned at different resolutions (we provide files at 200Kb resolution) using read start as a read position. No normalization was applied to the data.
Genome_build: mm9 or hg19
Supplementary_files_format_and_content: List of all interactions at single contact, 40Kb or 200 Kb resolution
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| Submission date |
Apr 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ilya M. Flyamer |
| E-mail(s) |
flyamer@gmail.com
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| Organization name |
Friedrich Miescher Institute for Biomedical Research
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| Lab |
Giorgetti
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE80006 |
Single-cell Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition |
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| Relations |
| BioSample |
SAMN04622162 |
| SRA |
SRX1685420 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2110077_182_pronucleus-w-o-inh-male-200kb.csv.gz |
26.0 Kb |
(ftp)(http) |
CSV |
| GSM2110077_182_pronucleus-w-o-inh-male-40kb.csv.gz |
34.8 Kb |
(ftp)(http) |
CSV |
| GSM2110077_182_pronucleus-w-o-inh-male-reads.csv.gz |
54.4 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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