R-Loops: Methods and Protocols(2528 Methods in Molecular Biology)

This detailed book compiles a series of laboratory protocols covering the most important aspects of R-loop biology. Beginning with a range of methods allowing 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 nucleotides in the R-loop context, as well as a number of specific protocols devoted to the investigation of R-loop topology and their functional roles in the biology of mitochondria and telomeres. Finally, a large block of chapters is dedicated to different methods allowing genome-wide mapping of DNA-RNA hybrids in various organisms. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, R-Loops: Methods and Protocols serves as an ideal resource for those working on R-loop homeostasis but also to scientists studying such areas of molecular and cell biology as genome integrity, DNA replication and repair, chromatin remodeling, transcription, RNA processing, modification and export, as well as for researchers elucidating the molecular mechanisms of cancer and genetic diseases.

Table of Contents:
Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy.- Detection of R-Loop Structures by Immunofluorescence Using the S9.6 Monoclonal Antibody.- R-Loop Detection in Bacteria.- Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in Saccharomyces cerevisiae.- Identification of RNA-DNA Hybrids Associated with R-Loops at the IgH Switch Sequence in Activated B Cells.- Locus-Specific Analysis of Replication Dynamics and Detection of DNA:RNA Hybrids by Immuno Electron Microscopy.- RNase H1, the Gold Standard for R-Loop Detection.- RNase H1 Hybrid-Binding Domain-Based Tools for Cellular Biology Studies of DNA-RNA Hybrids in Mammalian Cells.- Detection and Quantification of RNA Modifications on RNA/DNA Hybrids Using SID–UPLC–MS/MS.- DNA:RNA Hybrids at Telomeres in Budding Yeast.- Detection of TERRA R-Loops at Human Telomeres.- R-Loops and Mitochondrial DNA Metabolism.- Highly-Purified Top1-Bound DNA Fragments.- R-Loop Immunoprecipitation: A Method to Detect R-Loop Interacting Factors In Vivo.- Studies on Protein–RNA:DNA Hybrid Interactions by Microscale Thermophoresis (MST).- Studying R-Loop Recognizing Proteins Using Single-Molecule DNA Curtain Technique and Electrophoretic Mobility Shift Assay.- Immunostaining and Protein Pull-Down of Methyl-5-Cytosine-Marked RNA:DNA Hybrids.- In Vitro Binding of GADD45A to RNA:DNA Hybrids.- Proximity Ligation Assay for Detection of R-Loop Complexes upon DNA Damage.- Biochemical Analysis of RNA-DNA Hybrid and R-Loop Unwinding via Motor Proteins.- Topology of RNA:DNA Hybrids and R-Loops in Yeast.- Detecting and Mapping N6-Methyladenosine on RNA/DNA Hybrids.- Genome-Wide Native R-Loop Profiling by R-Loop Cleavage Under Targets and Tagmentation (R-Loop CUT&Tag).- Mapping R-Loops Using Catalytically Inactive RNaseH1 (R-ChIP).- Targeted Nuclease Approaches for Mapping Native R-Loops.- Quantitative DNA-RNA Immunoprecipitation-Sequencing with Spike-Ins.- DNA:RNA Immunoprecipitation from S. pombe Cells for qPCR and Genome-Wide Sequencing.- Genome-Wide Analysis of DNA-RNA Hybrids in Yeast by DRIPc-Seq and DRIP-Seq.- Quantitative, Convenient, and Efficient Genome-Wide R-Loop Profiling by ssDRIP-Seq in Multiple Organisms.