DRIP-seq

Abstract: R-loops are three-stranded nucleic acid structures that usually form during transcription and that may lead to gene regulation or genome instability. DRIP (DNA:RNA Immunoprecipitation)-seq techniques are widely used to map R-loops genome-wide providing insights into R-loop biology. However, annotation of DRIP-seq peaks to genes can be a tricky step, due to the lack of strand information when using the common basic DRIP technique. Here, we introduce DRIP-seq Optimized Peak Annotator (DROPA), a new tool for gene annotation of R-loop peaks based on gene expression information. DROPA allows a full customization of annotation options, ranging from the choice of reference datasets to gene feature definitions. DROPA allows to assign R-loop peaks to the DNA template strand in gene body with a false positive rate of less than 7%. A comparison of DROPA performance with three widely used annotation tools show that it identifies less false positive annotations than the others. DROPA is a fully customizable peak-annotation tool optimized for co-transcriptional DRIP-seq peaks, which allows a finest gene annotation based on gene expression information. Its output can easily be integrated into pipelines to perform downstream analyses, while useful and informative summary plots and statistical enrichment tests can be produced.

Abstract: R-loops are non-canonical nucleic structures composed of an RNA-DNA hybrid and a displaced ssDNA. Originally identified as a source of genomic instability, R-loops have been shown over the last decade to be involved in the targeting of proteins and to be associated with different histone modifications, suggesting a regulatory function. In addition, R-loops have been demonstrated to form differentially during the development of different tissues in plants and to be associated with diseases in mammals. Here, we provide a single-strand DRIP-seq protocol to identify R-loop-forming sequences in Drosophila melanogaster embryos and tissue culture cells. This protocol differs from earlier DRIP protocols in the fragmentation step. Sonication, unlike restriction enzymes, generates a homogeneous and highly reproducible nucleic acid fragment pool. In addition, it allows the use of this protocol in any organism with minimal optimization. This protocol integrates several steps from published protocols to identify R-loop-forming sequences with high stringency, suitable for de novo characterization. Graphic abstract: Figure 1.Overview of the strand-specific DRIP-seq protocol.

Abstract: R-loops are physiologically occurring structures in the genome that composed of a DNA/RNA hybrid and a displaced single-stranded DNA. R-loops have been observed in various organisms and shown to play important roles in regulating gene expression, DNA replication, genome stability, and other functions. The recent introduction of the protocol of DNA-RNA Immune-Precipitation (DRIP) followed by next-generation sequencing further propels the data accumulation of R-loop formation in different cellular contexts. In this study, we presented a user-friendly tool, DRIPer, for investigating DRIP-seq data to compare against a collection of publicly available DRIP-seq data and ENCODE ChIP-seq. Such comparisons allow correlation analysis and Kolmogorov-Smirnov tests to study associations via a given gene set, which could be for specific biological pathways, ontological functions, or other co-regulated genes. This powerful method will enable biologists to quickly evaluate the relationship of R-loops to nearby binding protein sites and target gene expression.