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

Preface
Contents
Contributors
Chapter 1: Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy
	1 Introduction
	2 Materials
		2.1 Genomic DNA Extraction
		2.2 DNA Digestion
		2.3 Native DNA Spreading by the ``BAC Method´´
		2.4 Automated Image Acquisition
		2.5 Image Annotation and Image Stitching
		2.6 DNA Content Quantification
		2.7 Image Analysis and Interpretation
	3 Methods
		3.1 Genomic DNA Extraction
		3.2 DNA Digestion
		3.3 Native DNA Spreading by the ``BAC Method´´
		3.4 Automated Image Acquisition
		3.5 Image Annotation and Image Stitching
		3.6 DNA Content Quantification
		3.7 Image Analysis and Interpretation
	4 Notes
	References
Chapter 2: Detection of R-Loop Structures by Immunofluorescence Using the S9.6 Monoclonal Antibody
	1 Introduction
	2 Materials
	3 Methods
		3.1 Grow the Cells on Coverslips and Fixation
		3.2 Quenching, Permeabilization, and Primary Antibody
		3.3 Secondary Antibody, DAPI Staining and Mounting
		3.4 Mounting and Microscopy
	4 Notes
	References
Chapter 3: R-Loop Detection in Bacteria
	1 Introduction
	2 Materials
		2.1 Genomic DNA Extraction
		2.2 Samples Preparation and Dot-Blotting
		2.3 Signal Revelation
	3 Methods
		3.1 Genomic DNA Extraction
		3.2 Samples Preparation and Dot-Blotting
		3.3 Signal Revelation
	4 Notes
	References
Chapter 4: Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in Saccharomyces cerevisiae
	1 Introduction
	2 Materials
		2.1 Detection of R-Loops by AID Overexpression
		2.2 Detection of R-Loops by Bisulfite Deamination
			2.2.1 DNA Extraction by CTAB Procedure
			2.2.2 DNA Digestion
			2.2.3 Bisulfite Treatment
			2.2.4 PCR Amplification
			2.2.5 Cloning and Sequencing
	3 Methods
		3.1 Detection of R-Loops by AID Overexpression
			3.1.1 Determination of Recombination Frequencies by Plating in Selective Media
			3.1.2 Determination of Recombination Frequencies by Flow Cytometry
			3.1.3 Detection of Rad52 Foci
		3.2 Detection of R-Loops by Bisulfite Deamination
			3.2.1 DNA Extraction by the CTAB Procedure
			3.2.2 DNA Digestion
			3.2.3 Bisulfite Treatment
			3.2.4 PCR Amplification
			3.2.5 Cloning and Sequencing
	4 Notes
	References
Chapter 5: Identification of RNA-DNA Hybrids Associated with R-Loops at the IgH Switch Sequence in Activated B Cells
	1 Introduction
	2 Materials
		2.1 DRIP-Seq
		2.2 Nondenaturing Sodium Bisulfite Probing
	3 Methods
		3.1 DNA-RNA Hybrid Immunoprecipitation and Sequencing
			3.1.1 Nucleic Acids Extraction from Activated B Cells
			3.1.2 DNA-RNA Hybrids Immunoprecipitation
			3.1.3 Library Preparation
			3.1.4 Sequencing of DRIP Libraries
		3.2 Nondenaturing Sodium Bisulfite Probing
	4 Notes
	References
Chapter 6: Locus-Specific Analysis of Replication Dynamics and Detection of DNA-RNA Hybrids by Immuno Electron Microscopy
	1 Introduction
	2 Materials
		2.1 Embedding and Lysis of Bacteria
		2.2 Extensive Genomic DNA Digestion
		2.3 Isolation of a Single Genomic DNA Locus from the Bulk Genomic DNA by Electrophoresis
		2.4 Preparation of R-Loop Carrying Spike-in  DNA
		2.5 RNase H Treatment
		2.6 Immuno DNA Spreading
		2.7 Image Acquisition
		2.8 Analysis and Interpretation
	3 Methods
		3.1 Embedding and Lysis of Bacteria
		3.2 Extensive Genomic DNA Digestion
		3.3 Isolation of Single DNA Loci from the Bulk Genomic DNA by Electrophoresis
		3.4 Preparation of R-Loop Carrying Spike-in  DNA
		3.5 RNase H Treatment (See Note 25)
		3.6 Immuno DNA Spreading
		3.7 Image Acquisition
		3.8 Analysis and Interpretation
	4 Notes
	References
Chapter 7: RNase H1, the Gold Standard for R-Loop Detection
	1 Introduction
	2 Using RNase H1 as a Reporter for the Detection of R-Loops
	3 Defects of Transcription and mRNA Processing Factors Lead to R-Loop Accumulation and DNA Damage
	4 R-Loops as Regulators of Transcription
	5 R-Loop Induced DNA Damage Due to Transcription-Replication Collisions
	6 Increase TERRA R-Loops in Telomeric Disfunction
	7 RPA as Sensor of R-Loops and Initiator of DNA Damage Response
	8 R-Loops and Chromatin Remodeling
	9 Detection and Quantification of R-Loops by RNase H1
	10 Is Overexpression of RNase H1 a Valid Tool to Study R-Loops? Benefits and Limitations
	References
Chapter 8: RNase H1 Hybrid-Binding Domain-Based Tools for Cellular Biology Studies of DNA-RNA Hybrids in Mammalian Cells
	1 Introduction
	2 Materials
		2.1 Immunofluorescence Analysis of DNA-RNA Hybrids Using HBD-GFP
		2.2 Live Cell Microscopy Analysis of HBD-GFP Recruitment to Microirradiation-Induced DNA Damage
	3 Methods
		3.1 Immunofluorescence Analysis of DNA-RNA Hybrids Using HBD-GFP
		3.2 Live Cell Microscopy Analysis of HBD-GFP Recruitment to Microirradiation-Induced DNA Damage
	4 Notes
	References
Chapter 9: Detection and Quantification of RNA Modifications on RNA-DNA Hybrids Using SID-UPLC-MS/MS
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Total Nucleic Acid Extraction
		2.3 RNase H Treatment of the Samples
		2.4 Nucleic Acids Hydrolysis to Nucleosides
		2.5 SID-UPLC-MS/MS Analysis
		2.6 Preparation of the Mix of Internal Standards for MS Analysis
	3 Methods
		3.1 Cell Culture: Preparing Matrigel Coated Plates and Culturing hPSCs
		3.2 Preparing the Cell Culture for DNA Extraction
		3.3 Nucleic Acid Extraction from Cells
		3.4 Digestion to Determine the Percentage of DNA
		3.5 RNase H Treatment of the Samples
		3.6 Ultrafiltration
		3.7 Hydrolysis to Nucleosides
		3.8 Preparation of the Mix of Internal Standards for MS Analysis
		3.9 SID-UPLC-MS/MS Analysis
	4 Notes
	References
Chapter 10: DNA-RNA Hybrids at Telomeres in Budding Yeast
	1 Introduction
	2 Materials
		2.1 Yeast Strains
		2.2 Culture Growth
		2.3 Cross-Linking of Cell Cultures
		2.4 Cell Lysis
		2.5 Sonication
		2.6 Immunoprecipitation
		2.7 Verification of Sonication
		2.8 qPCR
	3 Methods
		3.1 Culture Growth
		3.2 Cross-Linking of Cell Cultures
		3.3 Cell Lysis
		3.4 Preparation of the Magnetic Beads (See Note 9)
		3.5 Sonication
		3.6 Immunoprecipitation
		3.7 DRIP qPCR
		3.8 Computational Analysis
	4 Notes
	References
Chapter 11: Detection of TERRA R-Loops at Human Telomeres
	1 Introduction
	2 Materials
		2.1 Buffers and Solutions
		2.2 Isolation of Nuclei and Sonication
		2.3 DNA-RNA Immunoprecipitation
		2.4 Dot Blot
		2.5 qPCR
	3 Methods
		3.1 Cells Harvesting and Isolation of Nucleic Acids
		3.2 Sonication with Covaris E220
		3.3 DNA-RNA Immunoprecipitation
		3.4 Dot Blot
		3.5 qPCR
			3.5.1 qPCR Data Analysis
	4 Notes
	References
Chapter 12: R-Loops and Mitochondrial DNA Metabolism
	1 Introduction
		1.1 Evidence for the Prominent, Specific, Mitochondrial R-Loop
		1.2 Aberrant R-Loops: An Exaggerated Threat to Mitochondrial Genomic Stability?
		1.3 RNA-DNA Hybrids Are Not Synonymous with R-Loops
		1.4 Adventitious RNA Hybridization to DNA During Extraction
		1.5 General Methods of RNA/DNA Analysis Applicable to Mitochondrial   DNA
	2 Materials
		2.1 Isolation of Mitochondria from Solid Tissue
		2.2 Sucrose Gradient Centrifugation
		2.3 Mitochondrial DNA Extraction
		2.4 Isolation of Mitochondria from Cells
		2.5 Nucleic Acid Restriction and Modification, and Agarose Gel Electrophoresis
		2.6 Transfer of mtDNA to Solid Support & Hybridization (See Note 3)
	3 Methods
		3.1 Cotranscriptional R-Loop Formation Assessed by Nascent RNA Labeling and S9.6 Antibody Detection (See Note 4)
		3.2 Extraction of Mitochondria from Mouse Liver (See Note 7)
		3.3 Mitochondrial DNA Extraction from Isolated Mitochondria (See Note 8)
		3.4 Isolation of Mitochondria and mtDNA from Cultured Cells (See Note 9)
		3.5 Total Cellular DNA Isolation from Mammalian Cultured Cells After Crosslinking
		3.6 Total Cellular DNA Isolation from Tissue Homogenates
		3.7 Inter-Strand Cross-Linking with Psoralen and UV Light
		3.8 Nucleic Acid Modifications and 1D and 2D-AGE
		3.9 Transfer to Solid Support
		3.10 Hybridization to Determine the Steady-State Level of R-Loops
		3.11 Evaluating Adventitious Hybridization
	4 Notes
	5 Concluding Remarks
	References
Chapter 13: Highly Purified Top1-Bound DNA Fragments
	1 Introduction
	2 Materials
		2.1 DNA Purification by Cesium Chloride Density Gradient
		2.2 Top1cc Purity Evaluation by Western Blot in Purified DNA Samples
		2.3 DNA Fragmentation and K+-SDS Precipitation of Top1cc
		2.4 Real-Time PCR Analyses of Recovered DNA Fragments (Top1cc)
	3 Methods
		3.1 DNA Purification by Cesium Chloride Density Gradient
		3.2 Evaluation of Top1cc Content of Purified DNA with Western Blotting
		3.3 Fragmentation and K+-SDS Precipitation of Top1cc
		3.4 Real-Time PCR Analyses of Recovered DNA Fragments (Top1cc)
	4 Notes
	References
Chapter 14: R-Loop Immunoprecipitation: A Method to Detect R-Loop Interacting Factors
	1 Introduction
	2 Materials
		2.1 Biological Materials
		2.2 Buffers
		2.3 Commercial Reagents
		2.4 Equipment
	3 Methods
		3.1 RNA/DNA Hybrid  IP
			3.1.1 Cell Culture (Timing ~30 min)
			3.1.2 Protein A Dynabeads Preparation (Timing ~15 min plus O/N Incubation)
			3.1.3 Preparation of Double-Stranded Competitors (Timing ~2 h 15 min)
			3.1.4 Conjugation of S9.6 Antibody to the Protein A Dynabeads (Timing ~2 h 15 min)
			3.1.5 Preparation of Nuclear Extracts (Timing ~2 h)
			3.1.6 Immunoprecipitation (Timing ~2.5 h)
			3.1.7 Washes (Timing ~1 h)
			3.1.8 Elution for Western Blot (Timing ~15 min)
			3.1.9 Elution for Slot Blot (Timing ~45 min)
			3.1.10 Western Blot (WB) to Detect RNA/DNA-Binding Proteins (Timing ~6 h)
			3.1.11 Slot Blot (SB) to Assess the Efficiency of S9.6 Pull-Down (Timing ~4 h)
		3.2 Specificity of R-Loop Interactors: RNA/DNA hybrid IP with RNase H Control
			3.2.1 Cell Culture for Endogenous Hybrids Extraction (Timing ~30 min)
			3.2.2 Preparation of Endogenous Hybrids (Timing ~5 h)
			3.2.3 Preparation of BSA-Blocked and Antibody-Bound Protein A Dynabeads (Timing ~4 h 15 min plus O/N Incubation)
			3.2.4 Cell Culture for R-Loop IP with RNase H Digestion (Timing ~1 h)
			3.2.5 Binding of Genomic RNA/DNA Hybrids to Conjugated Antibody-Protein A Dynabeads (Timing ~2 h 30 min)
			3.2.6 Preparation of Protein Extracts (Timing ~3 h)
			3.2.7 Immunoprecipitation (Timing ~3 h 30 min)
	4 Notes
	References
Chapter 15: Studies on Protein-RNA:DNA Hybrid Interactions by Microscale Thermophoresis (MST)
	1 Introduction
	2 Materials
		2.1 Materials for Protein Labeling and MST Experiment; Buffer Exchange
	3 Methods
		3.1 Purification of Full-Length YTHDF2 with N-Terminal His-Tag
		3.2 Preparation of Modified RNA and RNA:DNA Hybrid Substrates
		3.3 Preparation of Dye Solution and Labeling of YTHDF2 with NT-647 RED-Tris-NTA Dye
		3.4 Preparation of 1:1 Ligand Dilution Series of the RNA:DNA Hybrid Substrate
		3.5 Preparing the Complexes and Performing the MST Experiment
		3.6 Analyze MST Data for NTA-Labeled YTHDF2 Binding to RNA:DNA Hybrid Substrate
	4 Notes
	References
Chapter 16: Studying R-Loop Recognizing Proteins Using Single-Molecule DNA Curtain Technique and Electrophoretic Mobility Shif...
	1 Introduction
	2 Materials
		2.1 Preparation of R-Loop Containing Lambda DNA (λ-DNA)
		2.2 Fabrication and Cleaning of Slides Containing Nano-Trench Patterns
		2.3 Biotinylated Liposome Stock Making
		2.4 Single-Tethered DNA Curtain Assay for R-Loop
		2.5 Double-Tethered R-Loop DNA Curtain Assay
		2.6 EMSA of TonEBP for R-Loop and Other DNA Constructs
	3 Methods
		3.1 Preparation of R-Loop Containing λ-DNA
		3.2 Fabrication and Cleaning of Slides Containing Nano-Trench Patterns
		3.3 Biotinylated Liposome Stock Making
		3.4 Single-Tethered DNA Curtain Assay for R-Loop
		3.5 Double-Tethered R-Loop DNA Curtain Assay
		3.6 EMSA for TonEBP with R-Loop and DNA Constructs
	4 Notes
	References
Chapter 17: Immunostaining and Protein Pull-Down of Methyl-5-Cytosine-Marked RNA:DNA Hybrids
	1 Introduction
	2 Materials
		2.1 Immunostaining of m5C Marked RNA:DNA Hybrids
		2.2 Biotin Pull-Down of m5C Marked RNA:DNA Hybrids
	3 Methods
		3.1 Immunostaining of m5C Marked RNA:DNA Hybrids
		3.2 Biotin Pull-Down of m5C Marked RNA:DNA Hybrids
	4 Notes
	References
Chapter 18: In Vitro Binding of GADD45A to RNA:DNA Hybrids
	1 Introduction
	2 Materials
		2.1 General Reagents
		2.2 Cell Culture and Related Equipment
		2.3 Agarose Gel Electrophoresis
		2.4 Reagents for the Binding Reactions
		2.5 Quantification
	3 Methods
		3.1 Labeling of RNA Oligonucleotides
		3.2 Generation of RNA:DNA Hybrid or R-Loops by Annealing Oligonucleotides
		3.3 Purification of GADD45A, PTB, and GFP
		3.4 Quantification of GADD45A, PTB and GFP Amounts Using Coomassie Staining
		3.5 R-Loop Pulldown Assay of GADD45A, PTB and GFP
		3.6 EMSA for GADD45A Binding to R-Loop and RNA:DNA Hybrid
	4 Notes
	References
Chapter 19: Proximity Ligation Assay for Detection of R-Loop Complexes upon DNA Damage
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
		2.3 Solutions
	3 Methods
		3.1 Reverse Transfection with RNaseH1 Plasmids
		3.2 Cover Glass Preparation
		3.3 Irradiation and Proximity Ligation Assay
		3.4 Proximity Ligation Assay
	4 Notes
	References
Chapter 20: Biochemical Analysis of RNA-DNA Hybrid and R-Loop Unwinding Via Motor Proteins
	1 Introduction
	2 Materials
		2.1 DNA and RNA Oligonucleotides
		2.2 Purified Proteins (See Note 1)
		2.3 Enzymes/Reaction Buffers (Purchased)
		2.4 Chemicals and Reagents
		2.5 Apparatus
	3 Methods
		3.1 Labeling of 5′-OH of DNA and RNA Oligonucleotide with γ-32P-ATP
		3.2 Generation of RNA-DNA and R-Loop Substrates
		3.3 Nucleic Acid Unwinding Reactions
		3.4 Native Gel Electrophoresis
	4 Notes
	References
Chapter 21: Topology of RNA:DNA Hybrids and R-Loops in Yeast
	1 Introduction
	2 Materials
		2.1 Preparation of S9.6 Magnetic Beads
		2.2 Crosslinking and Cells Collection
		2.3 Cell Lysis and Chromatin Extract
		2.4 Bead Washing and Crosslink Reversal
		2.5 DNA Purification and Library Amplification
		2.6 DNA Digestion and End Labeling
		2.7 Microarray Hybridization
	3 Methods
		3.1 Preparation of S9.6 Antibody Beads
		3.2 Crosslinking and Cells Collection
		3.3 Cell Lysis and Chromatin Extract Preparation for Immunoprecipitation
		3.4 Bead Washing and Crosslink Reversal
		3.5 DNA Purification and Library Amplification
		3.6 DNA Digestion and End Labeling
		3.7 Microarray Hybridization
		3.8 Data Processing and Visualization
	4 Notes
	References
Chapter 22: Detecting and Mapping N6-Methyladenosine on RNA/DNA Hybrids
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Immunostaining of m6A-Marked RNA/DNA Hybrids
		2.3 m6A-DIP
	3 Methods
		3.1 Immunochemical Detection of m6A-Marked RNA/DNA Hybrids
			3.1.1 hPSC Culture
			3.1.2 Preparation of the Slides for Immunostaining: Fixation and Permeabilization of the Cells
			3.1.3 Pretreatment of the Samples with RNases A and H or DNase I, and Depurination of DNA
			3.1.4 Co-immunostaining Cells for m6A and 5hmC
		3.2 m6A-DIP for Mapping the m6A-Marked RNA/DNA Hybrids
			3.2.1 Extraction of Genomic DNA from hPSCs
			3.2.2 Sonication of Genomic DNA
			3.2.3 RNase H Treatment
			3.2.4 Immunoprecipitation
	4 Notes
	References
Chapter 23: Genome-Wide Native R-Loop Profiling by R-Loop Cleavage Under Targets and Tagmentation (R-Loop CUT&Tag)
	1 Introduction
	2 Materials
		2.1 Recombinant pA-Tn5 and pA-Tn5 Transposome Assembly
		2.2 Purification of Recombinant GST-His6-2 x HBD Protein
		2.3 Cell Preparation and Binding Cells to Concanavalin A-Coated Beads
		2.4 Incubation with DNA-RNA Hybrid Sensors and Secondary Antibody
		2.5 Incubation with pA-Tn5 Adapter Complex and Tagmentation
		2.6 R-Loop CUT&Tag Library Preparation
		2.7 DNA Sequencing and Data Analysis
	3 Methods
		3.1 Recombinant pA-Tn5 and pA-Tn5 Transposome Assembly
		3.2 Purification of Recombinant GST-His6-2 x HBD Protein
		3.3 Cell Preparation and Binding Cells to Concanavalin A-Coated Beads
		3.4 Incubation with DNA-RNA Hybrid Sensors and Secondary Antibody
		3.5 Incubation with pA-Tn5 Adapter Complex and Tagmentation
		3.6 R-Loop CUT&Tag Library Preparation
		3.7 DNA Sequencing and Data Analysis
	4 Notes
	References
Chapter 24: Mapping R-Loops Using Catalytically Inactive RNaseH1 (R-ChIP)
	1 Introduction
	2 Materials
		2.1 Crosslinking and Sonication
		2.2 Chromatin Immunoprecipitation
		2.3 Library Construction and Sequencing
		2.4 General Supplies and Equipment
	3 Methods
		3.1 Crosslinking and Sonication
		3.2 Immunoprecipitation
		3.3 Sequencing Library Construction
	4 Notes
	References
Chapter 25: Targeted Nuclease Approaches for Mapping Native R-Loops
	1 Introduction
	2 Materials
		2.1 R-Loop Isolation (MapR)
		2.2 Bisulfite Conversion and Second-Strand Synthesis
	3 Methods
		3.1 R-Loop Isolation (MapR)
		3.2 Bisulfite Conversion and Second-Strand Synthesis
	4 Notes
	References
Chapter 26: Quantitative DNA-RNA Immunoprecipitation Sequencing with Spike-Ins
	1 Introduction
		1.1 RNA-DNA Hybrid Sequencing
		1.2 Potential Inaccuracies in the Normalization of Sequencing  Data
		1.3 The Use of Spike-In Controls in RNA-DNA Hybrid Sequencing Approaches
	2 Materials
		2.1 Selecting Regions for Spike-In Controls from E. coli
		2.2 Isolation of Genomic DNA from E. coli
		2.3 Amplification and Purification of PCR Fragments from E. coli Genomic  DNA
		2.4 In Vitro Transcription to Obtain Complementary  RNA
		2.5 Annealing and Purification of Synthetic RNA-DNA Hybrids
		2.6 Drosophila Schneider Cell Culture
		2.7 DNA-RNA Immunoprecipitation (DRIP)
		2.8 Quantitative PCR on Genomic and Spike-In RNA-DNA Hybrids (DRIP-qPCR)
		2.9 Library Preparation from ssDNA of RNA-DNA Hybrids
	3 Methods
		3.1 Preparation of Synthetic RNA-DNA Hybrid Spike-Ins from E. coli
			3.1.1 Spike-In Design
			3.1.2 Isolation of Genomic DNA from E. coli
			3.1.3 Amplification and Purification of PCR Fragments from E. coli Genomic  DNA
			3.1.4 In Vitro Transcription to Obtain Complementary  RNA
			3.1.5 Annealing and Purification of Synthetic RNA-DNA Hybrids
		3.2 Drosophila S2 Cells as Cell-Based Spike-Ins
		3.3 DNA-RNA Immunoprecipitation (DRIP)
		3.4 Quantitative PCR on Genomic and Spike-In RNA-DNA Hybrids (DRIP-qPCR)
		3.5 Library Construction from ssDNA of RNA-DNA Hybrids
		3.6 Bioinformatic Analysis and Normalization with Spike-In Counts
			3.6.1 Read Trimming
			3.6.2 Making Chimeric Genome Files for Mapping
			3.6.3 Normalization Using Synthetic Spike-Ins
			3.6.4 Normalization Using Cell-Based Spike-Ins
			3.6.5 Applications of Normalization Factors from Spike-Ins
	4 Notes
	References
Chapter 27: DNA:RNA Immunoprecipitation from S. pombe Cells for qPCR and Genome-Wide Sequencing
	1 Introduction
	2 Materials
		2.1 Yeast Culture
		2.2 gDNA Extraction and Purification
		2.3 gDNA Quantification by qPCR
		2.4 Shearing the  gDNA
		2.5 Immunoprecipitation
		2.6 Quantification of DRIP Signals by qPCR
		2.7 Equipment
	3 Methods
		3.1 Cell Culture
		3.2 Spheroplasting
		3.3 Cell Lysis
		3.4 Nucleic Acids Precipitation
		3.5 Confirm the Integrity of the  gDNA
		3.6 Shearing the  gDNA
			3.6.1 Option #1: Shearing the gDNA Using Sonication
			3.6.2 Option #2: Shearing the gDNA Using Restriction Enzymes
		3.7 Precipitation of the Sheared  gDNA
		3.8 Check the Quality of the Shearing
		3.9 gDNA Quantification by Quantitative PCR (qPCR)
		3.10 DNA:RNA Immunoprecipitation
			3.10.1 Preparation of Antibody-Coupled Magnetic Beads
			3.10.2 Immunoprecipitation and Washes
			3.10.3 DNA Purification
			3.10.4 Quantification of DRIP Signals by qPCR
			3.10.5 Strand-Specific Libraries
	4 Notes
	References
Chapter 28: Genome-Wide Analysis of DNA-RNA Hybrids in Yeast by DRIPc-Seq and DRIP-Seq
	1 Introduction
	2 Materials
		2.1 DNA-RNA Immunoprecipitation for Genome Wide in Yeast Cultures
		2.2 DRIP-seq and DRIPc-seq Data Alignment, Peak Calling, Annotation, and Visualization
	3 Methods
		3.1 DNA-RNA Immunoprecipitation in Yeast Cultures
		3.2 DRIP-seq and DRIPc-seq Data Alignment, Peak Calling, Annotation, and Visualization
	4 Notes
	References
Chapter 29: Quantitative, Convenient, and Efficient Genome-Wide R-Loop Profiling by ssDRIP-Seq in Multiple Organisms
	1 Introduction
	2 Materials
		2.1 General Reagents
		2.2 Buffers
		2.3 Equipment
		2.4 Software
	3 Methods
		3.1 Nuclei Isolation
		3.2 gDNA Extraction
		3.3 Digestion Using Cocktail of Restriction Enzymes
		3.4 DRIP
		3.5 Validation of Immunoprecipitation by qPCR
		3.6 ssDNA Library Preparation
		3.7 Additional Methods
		3.8 Data Analysis
	4 Notes
	5 Troubleshooting
	References
Index