R-loops are functional structures in chromatin comprising one single-stranded DNA and a DNA:RNA hybrid. Here, we report ssDRIP-seq, a single-strand DNA ligation-based library preparation technique for genome-wide identification of R-loops. When applied in Arabidopsis, ssDRIP-seq exhibits high efficiency, low bias and strand specificity. We found that Arabidopsis R-loops are enriched by both AT and GC skews, and are formed in the sense and antisense orientations. R-loops are strongly enriched in gene promoters and gene bodies, and are highly associated with noncoding RNA and repetitive genomic regions. Furthermore, R-loops are negatively correlated with CG DNA hypermethylation, and are prevalent in regions with multiple chromatin modifications, showing strong correlations with the activated and repressed gene loci. Our analyses indicate that R-loops are common features in the Arabidopsis genome and suggest that the R-loops play diverse roles in genome organization and gene regulation, thereby providing insights into plant nuclear genome formation and function.
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We thank all members of the Sun and Yang labs for the useful discussion and suggestions, and we appreciate F. Chédin for sharing the detailed DRIP-seq protocol. We also appreciate the suggestions from three anonymous reviewers for improving the quality of this manuscript. This work was supported by grants from the National Key R&D Program (2016YFA0500800 to the Sun Lab), the National Natural Science Foundation of China (91540109 and 31671381 to X.Y.), the Tsinghua University Initiative Scientific Research Program (to the Sun Lab), the Tsinghua-Peking Joint Center for Life Sciences and the 1000 Young Talent Program of China (to Q.S. and X.Y.).
The authors declare no competing financial interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Supplementary Figures 1–11
Arabidopsis public data used in this study.
Information for software used in this study.
Detailed information of R-loop peaks and their overlap with various genome features.
Information for primers used in this study.
Identified R-loop motifs by HOMER. The files include HOMER Known Motif Enrichment Results, forward peak motifs, reverse peak motifs, and unstranded peak motifs.
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Science Advances (2019)
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