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| Status |
Public on Oct 18, 2023 |
| Title |
DNA mutant WGS-seq |
| Sample type |
SRA |
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| Source name |
Tail
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| Organism |
Mus musculus |
| Characteristics |
tissue: Tail
genotype: Padi6P620A/P620A
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| Growth protocol |
For the experiment, males of 8-12 weeks of age and females of 4-8 weeks of age were used. The day before the start of the experiment, the males were separated. On day 1, at 2 pm, intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) were given to the females. After approximately 52 hours, the same females received intraperitoneal injections of human chorionic gonadotropin (hCG). Male was sacrificed approximately 12 hours after the female hCG injection. The cauda epididymis and vasa deferentia were dissected and the spermatozoa were collected in the human tubal fluid medium (HTF) (EmbryoMax® HTF MR-070-D). Spermatozoa were counted and incubated at 37°C within a humidified atmosphere of 5% CO2 in air. Females were sacrificed approximately 13 hours after the hCG injection. Ampullae were placed in hyaluronidase 1mg/ml (H6254-500MG) drops to break down the oocyte-cumulus complex and collect MII oocyte. The oocytes were moved to the HTF medium and a 2x106 sperm/ml concentration of spermatozoa were added. After 4 - 6 hours of incubation at 37°C within a humidified atmosphere of 5% CO2 in air, the fertilised oocytes were washed, to remove the spermatozoa e debris, transferred in KSOM medium and incubated overnight at 37°C within a humidified atmosphere of 5% CO2 in air. The embryos' status was evaluated in the morning and the next 120 hours.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The presence of the Padi6 variant, the removal of Neo box and the presence of the flippase gene were demonstrated by PCR on tail DNA extracted with standard procedures. Amplicons of 430 bp and 290 bp were amplified from the mutant or wild type Padi6 alleles, respectively, by using the primers Padi6_RimF and Padi6_RimR. This difference was due to a fragment of the NEO box remaining after the flippase recombination. The presence of the mutation was also identified by digestion with the restriction enzyme StuI, which was generated by the mutation, after amplification of a 420 bp fragment with the primers For LA HindIII + SnaBI and Rev GEN Padi6. The presence of the NEO box was detected using the primers PGK1A For and Padi6_RimR. The presence of the flippase was detected using the primers FLP_F and FLP_R. The primer sequences are listed in the Supp. Table S17.
Whole genome sequencing was performed on genomic DNA of homozygous female. The DNA samples were sequenced 150 bp pair-end at BIODIVERSA srl Service (Milan, Italy) using the Nextera DNA Prep illumina and the Illumina NovaSeq6000 platform.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The read quality was checked using the FASTQC tools (Andrews S, 2010). Reads were aligned to the mouse genome reference assembly (mm39) using the BWAmem software package v0.7.17 (Li H, 2009). PCR duplicates were filtered out by Picard v2.9 (http://picard.source forge.net), and the Dellytools v1.1.6 with default parameters were employed to calculate the structural and CNV variants.
Assembly: mm39
Supplementary files format and content: VCF file for Svs
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| Submission date |
Oct 15, 2023 |
| Last update date |
Oct 18, 2023 |
| Contact name |
Francesco Cecere |
| E-mail(s) |
francesco.cecerengs@gmail.com
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| Phone |
3888903265
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| Organization name |
University of Campania "Luigi Vanvitelli"
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| Department |
DiStABiF
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| Lab |
Riccio
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| Street address |
Via A. Vivaldi, n. 43
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| City |
Caserta |
| State/province |
Caserta |
| ZIP/Postal code |
81100 |
| Country |
Italy |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE245425 |
A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes as well as failure of epigenetic reprogramming and zygotic genome activation in embryos [WGS] |
| GSE245426 |
A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes as well 2 as failure of epigenetic reprogramming and zygotic genome activation in embryos |
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| Relations |
| BioSample |
SAMN37821681 |
| SRA |
SRX22095458 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7842651_padi6.sv.vcf.gz |
6.8 Mb |
(ftp)(http) |
VCF |
SRA Run Selector |
| Raw data are available in SRA |
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