Titlebook: R-Loops; Methods and Protocol Andrés Aguilera,Alexey Ruzov Book 2022 The Editor(s) (if applicable) and The Author(s), under exclusive licen

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,Studies on Protein–RNA:DNA Hybrid Interactions by Microscale Thermophoresis (MST), of the interaction of the N-terminal his-tagged 6-methyladenine (m.A) reader protein YTHDF2 with m.A modified and unmodified RNA, in single-strand configuration or with RNA:DNA hybrid substrates. The described protocol is also suitable for studies of interactions with proteins binding to double-stranded RNA or DNA substrates.

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Book 2022allowing for the detection of DNA-RNA hybrids, as well as their purification and visualization by electron microscopy, the volume continues with methods based on the use of RNase H-derived tools to detect DNA-RNA hybrids .in vitro. and .in vivo.. Several protocols permit studying non-canonical RNA n

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Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in , genome integrity. Here, we describe the protocols used in the yeast . to infer the presence of R-loops through increased AID-induced DNA damage, measured as increased recombination or Rad52 foci formation as well as to detect single R-loop molecules and determine their length at particular genomic sites via bisulfite treatment and amplification.

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,RNase H1 Hybrid-Binding Domain-Based Tools for Cellular Biology Studies of DNA–RNA Hybrids in Mammanowledge about R loops and factors associated with their formation and removal. Here, we describe the use of fluorescently tagged HBD, the hybrid-binding domain of RNase H1, as a tool for analyzing DNA–RNA hybrids in different contexts using live-cell microscopy and immunofluorescence experiments.

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,DNA–RNA Hybrids at Telomeres in Budding Yeast,ect recovered material, and furthermore allows the precipitation of other proteins from the identical cross-linked material. Although both methods are successful in terms of detecting DNA–RNA hybrids at telomeres, the R-ChIP method yields an approximately ten-fold increased enrichment.