The vertical bars represent methylation (black portion) observed for the indicated 5′ CpG dinucleotides covered by at least ten sequence reads; the analysed region corresponds to the central and 3′ part of the 5′ LTR, as indicated in the IAP scheme and by CpG position. Two RNA-seq libraries from hamster oocytes, which were outliers in PCA and had poor quality/low complexity (clearly because of a limited amount of starting material), were excluded from analysis. Thin-layer chromatography showed only one peak of radioactivity corresponding to unaltered (99m)Tc-albumin nanocolloid. The genome was divided into 1 kb windows and, for each window, alignments of 24–31-nucleotide reads were counted with fractional counts.

featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Borchert W, Grady ST, Chen J, DeVille NV, Roscoe C, Chen F, Mita C, Holland I, Wilt GE, Hu CR, Mehta U, Nethery RC, Albert CM, Laden F, Hart JE. The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons.Average C t values of the technical replicates were normalized to the housekeeping genes Ma HPRT and Ma B2MG using the ΔΔ C t method 53. The animals used for experiments were euthanized by intraperitoneal injection of a lethal dose of Euthasol (Samohyl). All of the animal experiments were approved by the Animal Experimentation Committee at the RIKEN Tsukuba Institute (T2019-J004) and the Institutional Animal Use and Care Committee at the Institute of Molecular Genetics of the Czech Academy of Sciences (approval no. This is possible due to the unique promiscuity of hamster ova, which allows them to fuse with non-hamster sperm. The red and blue points depict genes of which the transcripts were present at significantly higher or lower levels in fully-grown Mov10l1 –/– oocytes, respectively.

A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR. Nuclei were stained with 1 μg ml −1 DAPI for 10 min, slides were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) and images were acquired using the DM6000 or Leica SP8 confocal microscope. Notably, Lypd6b and Kif5c, two of the three most upregulated genes are genomic neighbours, suggesting a common regulation of the locus, which might involve Kif5c 3′-UTR-derived piRNAs and/or derepression of retrotransposons in the first Lypd6b intron (Fig.Obligatory or true hibernation occurs when certain species become fat in the summer months and hibernate in the winter months. Ribosomal RNA (rRNA) was depleted from RNA used for transcriptome analysis using the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (Epicentre) or the QIAseq FastSelect–rRNA HMR Kit (Qiagen) according to the manufacturer’s protocols. Only regions covered by at least four fragments in Mov10l1 +/– and Mov10l1 –/– libraries were included in the analysis. Derepression of L1 retrotransposons was absent or the anti-ORF1 antibody did not cross-react with the hamster protein. piRNAs target retrotransposons during mouse oogenesis 17, 18, but the maternal piRNA pathway is partially redundant with RNA interference (RNAi) 19.

Next, to remove alignments arising from excessive PCR amplification, alignments were deduplicated using the deduplicate_bismark script. This experiment was performed once; the impact of the mutation on Mov10l1 expression in the testes was confirmed using RNA-seq analysis (Extended Data Fig. Dilution factors are presented in the form of a range because the optimal dilution is a function of many factors, such as antigen density, permeability, etc. Ubiquitination-deficient mutations in human Piwi cause male infertility by impairing histone-to-protamine exchange during spermiogenesis.

affiliations: names in common with same affiliation weighs toward the two names being the same person. Coloring of bars indicates proportions of miRNAs and small RNAs derived from repetitive sequences, mRNA, and other sequences. For transcriptome analyses, the DESeq2 package was used, which internally uses two-sided Wald test and corrects P values for multiple comparisons using the Benjamini–Hochberg method. At the same time, the hamster outclassed the mouse model for specific aspects of human biology 25, 26. While the loss of DNA methylation at unique loci would have escaped detection (including those identified in Piwil3 –/– hamster oocytes 34), bisulfite sequencing of Mov10l1 –/– oocytes yielded enough unique sequencing reads to examine global methylation levels of retrotransposon subfamilies (Extended Data Fig.

Heterozygotes were fertile and segregation of genotypes did not deviate significantly from the Mendelian ratio but homozygotes of both sexes were sterile (Supplementary Table 4). Based on immunoelectrophoresis and/or ELISA, the antibody reacts with whole molecule syrian hamster IgG. Mammalian primary piRNAs originate from specific loci (piRNA clusters) as long precursor transcripts that interact with the essential and conserved helicase MOV10L1, which feeds precursor transcripts into the piRNA biogenesis mechanism 3, 4, 5. Rehydrate with the indicated volume of dH2O (see product specification sheet) and centrifuge if not clear.The sample sizes and numbers of replicates are provided in the figure captions and, for RNA-seq analyses, in Supplementary Table 10. The Venn diagram shows overlap of upregulated DEGs (a single gene was common for downregulated DEGs). c, Testicular 24–32-nucleotide small RNAs mapping to LTR retrotransposon groups selected for low nucleotide exchange rate and high abundance of putative piRNAs (Extended Data Fig.