Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq

Célia Alecki; Nicole J Francis

doi:10.21769/BioProtoc.4011

Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq

Bio Protoc. 2021 May 5;11(9):e4011. doi: 10.21769/BioProtoc.4011.

Authors

Célia Alecki^{1

2}, Nicole J Francis^{1

2

3}

Affiliations

¹ Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC, H2W 1R7, Canada.
² Département de biochimie et médecine moléculaire Université de Montréal, 2900 Boulevard Edouard-Montpetit, Montréal, QC, H3T 1J4, Canada.
³ Division of Experimental Medicine, McGill University, 1001 Decarie Boulevard, Montreal, QC, H4A 3J1, Canada.

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.

Keywords: DRIP; Drosophila melanogaster; R-loop; RNA-DNA hybrid; S9.6 antibody; Tissue culture cells.