系统生物学

Chromatin-associated RNAs (caRNAs) have been increasingly recognized as key regulators of gene expression and genome architecture. A few technologies, such as ChRD-PET and RedChIP, have emerged to assess protein-mediated RNA–chromatin interactions, but each has limitations. Here, we describe the TaDRIM-seq (targeted DNA-associated RNA and RNA–RNA interaction mapping by sequencing) technique, which combines Protein G (PG)-Tn5-targeted DNA tagmentation with in situ proximity ligation to simultaneously profile caRNAs across genomic regions and capture global RNA–RNA interactions within intact nuclei. This approach reduces the required cell input, shortens the experimental duration compared to existing protocols, and is applicable to both mammalian and plant systems.

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