corresponding to positions 11−45 of piRNA loci (U[T]:G:C:A = 25.7:23
.4:21.8:29.1)^43. For Fig. 4f, 27-nt genomic sequence pools correspond-
ing to positions 19−45 nt of 1,946 Siwi-dominant and 1,259 BmAgo3-
dominant piRNA loci (Extended Data Fig. 3a) were extracted and used
to calculate the similarity score between the weighted BmZuc motif
for Siwi or BmAgo3 and each extracted genomic sequence from the
Siwi- or BmAgo3-dominant piRNA loci by a sliding window approach
(Extended Data Fig. 5g, lower).
Statistics and reproducibility
Experiments in Figs. 1a, 3c−f, Extended Data Figs. 2d−g, 4d, f were inde-
pendently performed twice with similar results. Experiments in Figs.
2 b, c, 3g−i, 4d and Extended Data Figs. 2b, h, 3i, 4b, c, 5c were performed
once. For the statistical analyses in Figs. 2a, 3l, Extended Data Figs. 3f, 5i,
detailed statistical values were summarized in Supplementary Table 2.
To estimate of effect sizes in Fig. 3l, the cohensD function in the lsr
package was used. To estimate of effect sizes in Fig. 2a and Extended
Data Figs. 3f, 5i the wilcoxsign_test and wilcox_test function in the coin
package was used. No statistical methods were used to predetermine
sample size. All experiments were not randomized and no blinding was
used during data analysis.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
The sequencing data reported in this paper are publicly available in
DDBJ, under the accession number DRA008549. All other data are avail-
able from the authors upon reasonable request.
Code availability
All code required for bioinformatics analysis in this paper is available
at https://github.com/kshoji-nt/BmZuc_cleavage.
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Acknowledgements We thank T. Kusakabe and T. Tatsuke for providing BmArmi expression
vectors, K. Förstemann for providing hCas9 and sgRNA expression vectors, T. Kiuchi for
sharing unpublished data and helpful discussion, and K. Kiyokawa and T. Horiuchi for technical
assistance. A part of Illumina sequencing was performed in the Vincent J. Coates Genomics
Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation
Grant. We also thank Life Science Editors for editorial assistance, and P. Zamore and members
of the Tomari laboratory for critical comments on the manuscript. This work was in part
supported by a Grant-in-Aids for Scientific Research on Innovative Areas (grant 26113007 to
Y.T.) from the Ministry of Education, Culture, Sports, Science and Technology in Japan and a
Grant-in-Aid for Scientific Research (S) (grant 18H05271 to Y.T.), Grant-in-Aid for Scientific
Research (B) (grant 16KT0064 to Y.S. and S.K.), Grant-in-Aid for Scientific Research on
Innovative Areas (grant 17H06431 to S.K.), Grant-in-Aid for Young Scientists (B) (grant 17K17673
to N.I.), Grant-in-Aid for Scientific Research (C) (grant 19K06484 to N.I.), and a Grant-in-Aid for
JSPS Fellows (grant 17J02408 to K.S.).
Author contributions N.I., K.S. and Y.T. conceived and designed the experiments and wrote the
manuscript. N.I. performed biochemical experiments. K.S. performed bioinformatics analyses.
Y.S. and S.K. supervised the bioinformatics analyses. Y.T. supervised the research. All the
authors discussed the results and approved the manuscript.Competing interests The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
1966-9.
Correspondence and requests for materials should be addressed to Y.T.
Peer review information Nature thanks René Ketting and the other, anonymous, reviewer(s) for
their contribution to the peer review of this work.
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