数字农科院2.0

mDRIP-seq is a high-throughput method for quantitative profiling of R-loop landscape

文献类型： 外文期刊

作者： Changbin Sun; Zhenzhen Wang; Qin Li; Qianwen Sun; Wei Xu

关键词： N/A

期刊名称： Science Bulletin

ISSN： 2095-9273

年卷期： 2025 年

页码：

收录情况： SCIE（2025版） ; ; EI（2025版） ; ; CSCD（2025-2026年度） ; ; 科技核心（2024版）

摘要： R-loops are three-stranded nucleic acid structures, in which RNA hybridizes to one DNA strand and forces another DNA strand to bulge out. Accumulating evidence demonstrated that R-loops play important roles in many physiological processes, including regulation of gene expression, telomere stability, class-switch recombination, and mitochondrial DNA replication. However, unscheduled R-loops can be formed as genomic threats to trigger DNA replication stress and genome instability, which may contribute to neurodegeneration, cancer, and inflammatory diseases. Given the versatile roles of R-loops in cellular processes, it is essential to precisely map their genome-wide distribution and quantify their dynamics for various cell types, tissues, and organisms under different conditions and developmental stages to address their sources, roles, and resolution. In this study, we developed a multi-sample pooling DNA:RNA ImmunoPrecipitation followed by sequencing (mDRIP-seq) method for high-throughput and quantitative profiling of R-loops. Different from existing methods, mDRIP-seq combines a barcoding strategy to pool multiple samples together for DRIP and library preparation with strand-specific sequencing. Considering that there is still a lack of methods for barcoding intact DNA:RNA hybrids, we established a tailing and single-strand ligation method that utilizes terminal deoxynucleotidyl transferase (TdT) to tail the 3′ terminus of DNA in DNA:RNA hybrids and a short splint to facilitate ligation of the barcoded adapter to the tailed hybrids.

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