|
| Status |
Public on Jul 02, 2021 |
| Title |
PIWIL3homo - smRNA-seq_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
PIWIL3homo - smRNA-seq
|
| Organism |
Mesocricetus auratus |
| Characteristics |
tissue: MII oocyte
genotype: m/m
|
| Treatment protocol |
PIWIL1 and PIWIL3 mutant golden hamster was generated and established using CRISPR/Cas9 system. See reference for details (Hasuwa et al.).
|
| Growth protocol |
Golden hamsters were purchased from Japan SLC and maintained with 14 light and 10 dark cycle. All animal experiments were approved by the Animal Care and Use Committee of the Keio university.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mature females were induced to superovulate by i.p. injection of 40 IU PMSG (ASKA Animal Health) at 1200 to 1400 on the day of conspicuous, postestrus vaginal discharge (day 1 of the estrous cycle). GV oocytes were collected from ovary after 102h PMSG injection. MII oocytes were collected from oviduct after 115h PMSG injection. PN zygotes were collected from oviduct after 91 or 115h PMSG injection followed by mating with male during the night of day4 or 5 (depend on the female estrus cycle).
small-RNA-seq: Total RNAs for small RNA sequence were extracted from MII oocytes of PIWIL1 and PIWIL3 homozygous and heterozygous mutants. The construction of small RNA libraries was performed using NEXTFLEX® Small RNA-Seq Kit v3 (PerkinElmer) with the manufacturer’s instructions.
RNA-seq: Total RNA for RNA-seq were purified from 50 MII oocytes of 2 to 4-month-old golden hamsters using NucleoSpin RNA Plus XS (MACHEREY-NAGEL). RNA-seq libraries were construct using total RNA from MII oocytes and SMART-Seq Stranded Kit (TaKaRa Bio).
Bisulfite-seq: DNA from thirty GV oocytes were spiked with 1% unmethylated lambda phage DNA. Libraries were generated by post bisulfite adaptor tagging method with 8 cycles of library amplification.
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| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
PIWIL3piRNA_count.tsv
|
| Data processing |
small-RNA-seq: The adapter sequence and 4nt random sequences at 3’ and 5’ ends were removed cutadapt v1.14. A quality filter of 30 was adopted by removing sequences below this threshold. Reads at the range of 10 ~ 40nt after adapter-removal were used for further analysis. Reads were mapped to the hamster reference genome (hamster.sequel.draft-20200302.arrow.fasta; 3) using Bowtie (v1.1.1), permitting 0 mismatches, and genome-mapped reads were used for further analysis. Genome mapped reads were compared between replicate samples and combined due to their high reproducibility. Reads were further mapped to tRNA, rRNA, sn/snoRNA, and miRNA. The read count mapped to miRNAs were used to normalize small RNA reads obtained from heterozygous and homozygous mutants. Read counts mapped to each unique small RNA sequence were compared between heterozygous and homozygous samples, and those with an 4-fold decrease and identical to previously described PIWI-associated piRNAs were extracted as PIWIL1- or PIWIL3-piRNA.
RNA-seq: Low-quality sequence (a quality value lower than 30) and 3nt random sequence was removed using cutadapt software. Mapping to the golden hamster genome (hamster.sequel.draft-20200302.arrow.fasta; 3) was performed using HISAT2 using default parameters. featureCount v2.01 was used to count reads mapped to genes. featureCounts output was further processed using the TCC-GUI version 2019.02.08 using default parameters. The TCC-GUI uses the TMM normalization method, and edgeR is used for the filtering of differentially expressed genes (DEGs).
Bisulfite-seq: Reads were trimmed to remove low quality bases, and were mapped to the golden hamster genome using Bismark v0.20.0. Methylation data at CG and non-CG sites covered with 10-500 reads were extracted for the downstream analyses.
Genome_build: hamster.sequel.draft-20200302.arrow.fasta (newly sequenced genome available under accession number PRJDB10770 in DDBJ)
Supplementary_files_format_and_content: small-RNA-seq: each unique PIWIL1/PIWIL3-piRNA sequence (see reference for the defenition of PIWIL1/PIWIL3-piRNA) with count of obtained reads in tab separated format.
Supplementary_files_format_and_content: RNA-seq: expression level of genes in each samples calculated by using TCC-GUI (TMM normalization method and edgeR is used in TCC), in csv format.
Supplementary_files_format_and_content: Bisulfite-seq: position of CpG (name of scaffold, start position, and end position), methylation ratio, number of methylated CpG, and number on unmethylated CpG in tab separated format.
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| |
|
| Submission date |
Jan 06, 2021 |
| Last update date |
Jul 03, 2021 |
| Contact name |
Yuka W. Iwasaki |
| E-mail(s) |
iwasaki@keio.jp
|
| Organization name |
Keio University School of Medicine
|
| Department |
Department of Molecular Biology
|
| Lab |
Siomi Lab
|
| Street address |
35 Shinanomachi, Shinjuku-ku,
|
| City |
Tokyo |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL29575 |
| Series (1) |
| GSE164356 |
Production of functional oocytes requires maternally expressed PIWI genes and piRNAs in golden hamsters |
|
| Relations |
| BioSample |
SAMN17229216 |
| SRA |
SRX9794869 |
