Epigenomics: Methods and Protocols

Preface
Contents
Contributors
Part I: Analysis of the Epigenome: DNA Methylation
	Chapter 1: DNA Methylation Analysis Using Bisulfite Pyrosequencing
		1 Introduction
			1.1 Principles of Pyrosequencing Technology
			1.2 Principles of Bisulfite Conversion
			1.3 Overview of Bisulfite Pyrosequencing
		2 Materials
			2.1 Bisulfite Conversion
			2.2 PCR
			2.3 Pyrosequencing
		3 Methods
			3.1 Design and Validation of Primers for Pyrosequencing
			3.2 Bisulfite PCR for Pyrosequencing
			3.3 Bisulfite Pyrosequencing
				3.3.1 Pyrosequencer Preparation
				3.3.2 Preparation of Pyrosequencing Template
				3.3.3 Pyrosequencing Reaction
				3.3.4 Pyrosequencer Cleanup
				3.3.5 Data Analysis
		4 Notes
		References
	Chapter 2: Post-bisulfite Adaptor Tagging Based on an ssDNA Ligation Technique (tPBAT)
		1 Introduction
			1.1 Selection of the DNA Purification Method for tPBAT Library Preparation
			1.2 Sequencing Libraries of Highly Biased Nucleotide Composition
			1.3 Amplification of the Sequencing Library
		2 Materials
			2.1 Commercial Kits, Enzymes, and Consumables
			2.2 Solutions
			2.3 Oligonucleotides (OPC Grade)
		3 Methods
			3.1 Bisulfite Treatment
			3.2 Random Priming
			3.3 TACS Ligation
			3.4 Primer Extension (1)
			3.5 Primer Extension (2)
			3.6 Library QC
			3.7 Library Amplification
			3.8 Sequencing
		4 Notes
		References
	Chapter 3: Reduced Representation Bisulfite Sequencing (RRBS)
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Oligonucleotides
			2.3 Plastic Disposables
			2.4 Equipment
		3 Methods
			3.1 MspI Digestion of Genomic DNA (Day 1, 2.5 h)
			3.2 End Repair and dA-Tailing (Day 1, 2 h)
			3.3 Adaptor Ligation (Day 1, 1.5 h; Day 2, 1 h)
			3.4 Bisulfite Conversion of Adaptor-Ligated DNA (Day 2, 4 h)
			3.5 PCR Amplification (Day 2, 1 h; Day 3, 1 h)
			3.6 Library Quality Control (Day 3, 1 h, or Longer When the Second DNA Purification or an Additional PCR Amplification Is Requ...
			3.7 Sequencing
			3.8 Data Analysis (See Note 16)
		4 Notes
		References
Part II: Analysis of the Epigenome: Histone Modification and Chromatin Structure
	Chapter 4: Chromatin Immunoprecipitation Sequencing (ChIP-seq) for Detecting Histone Modifications and Modifiers
		1 Introduction
		2 Materials
			2.1 Chromatin Fixation and Fragmentation
			2.2 Immunoprecipitation
			2.3 DNA Purification
			2.4 NGS Library Prep
		3 Methods
			3.1 Preparation of Cell Suspension and Tissue Lysate
			3.2 Chromatin Fixation and Fragmentation
				3.2.1 Histone Modifications
				3.2.2 Histone Modifiers
			3.3 Immunoprecipitation
			3.4 DNA Purification
			3.5 NGS Library Prep
			3.6 Data Analyses
		4 Notes
		References
	Chapter 5: ATAC-Seq Analysis of Accessible Chromatin: From Experimental Steps to Data Analysis
		1 Introduction
		2 Materials
			2.1 Reagents for Preparation of Mouse Male Germ Cells
			2.2 Tn5 Transposase
			2.3 Other Reagents
			2.4 Buffers
			2.5 Equipment
			2.6 Software
			2.7 Reagent Setup
		3 Methods
			3.1 Preparation of Mouse Male Germ Cells
			3.2 Cell Lysis and Transposition
			3.3 DNA Purification
			3.4 PCR Amplification (Library Generation)
			3.5 Library Purification
			3.6 Assessing Library Quality and Sequencing
			3.7 ATAC-seq Alignment
			3.8 Confirmation of Reproducibility
			3.9 Data Visualization Through BAM Coverage Tracks
			3.10 Peak Calling with MACS2
			3.11 Data Visualization Using ngs.plot
			3.12 GREAT Analysis
			3.13 De Novo Motif Analysis
		4 Notes
		References
	Chapter 6: Low-Input CUT&RUN for Mouse Oocytes and Preimplantation Embryos
		1 Introduction
		2 Materials
			2.1 Reagents for Sampling of Oocytes and Preimplantation Embryos
			2.2 Reagents
			2.3 Buffers
		3 Methods
			3.1 Removal of Zona Pellucida and Polar Bodies (See Note 5)
			3.2 Bind Primary Antibody
			3.3 Bind pA-/pAG-MNase
			3.4 Prepare Magnetic Beads
			3.5 Digestion
			3.6 DNA Purification
			3.7 End Repair and Adaptor Ligation (NEBNext Ultra II DNA Library Prep Kit)
			3.8 Clean Up the Adaptor-Ligated DNA
			3.9 PCR Amplification and Library Purification
		4 Notes
		References
	Chapter 7: Imaging Chromatin Accessibility by Assay of Transposase-Accessible Chromatin with Visualization
		1 Introduction
		2 Materials
			2.1 Preparation of Recombinant Tn5
			2.2 ATAC-see
			2.3 Cell Culture
		3 Methods
			3.1 Preparation of Recombinant Tn5 Protein
			3.2 Preparation of Tn5 Complex
			3.3 Preparation of ATAC-see Reaction Mixture
			3.4 ATAC-see for Microscopic Observation
			3.5 ATAC-see Analysis for Flow Cytometry
		4 Notes
		References
	Chapter 8: STREAMING-Tag System: Technology to Enable Visualization of Transcriptional Activity and Subnuclear Localization of...
		1 Introduction
		2 Materials
			2.1 Cell Culture of Mouse Embryonic Stem Cells
			2.2 Multicolor Imaging of SNAPtag, mTetR, and MCP Spots
			2.3 Software and Modules/Packages for the Image Analysis
		3 Methods
			3.1 Cell Culture and Passage of Mouse Embryonic Stem Cells
			3.2 SNAPtag Labeling and Image Acquisition by Confocal Microscope
			3.3 Pre-Processing of Acquired Images
			3.4 Detection of Spots and Measurement of 2D Distances at Sub-Pixel Resolution
		4 Notes
		References
	Chapter 9: Bioinformatics Pipelines for Identification of Super-Enhancers and 3D Chromatin Contacts
		1 Introduction
		2 Materials
			2.1 Computer/Hardware
			2.2 Software
			2.3 NGS Data from Other Sources
		3 Methods
			3.1 Processing H3K27ac ChIP-seq Data
				3.1.1 ChIP-seq Alignment
				3.1.2 Data Visualization Through BAM Coverage Tracks
			3.2 Identification of Cell Type-Specific Enhancers and Super-Enhancers
				3.2.1 Peak Calling with MACS2 and Identification of Active Enhancers
				3.2.2 Super-Enhancer Calling
			3.3 Predicted Functions and Biological Significance of Super-Enhancers
				3.3.1 MAnorm Analysis
				3.3.2 GREAT Analysis
				3.3.3 De novo Motif Analysis
			3.4 Inferring Long-Range Super-Enhancer Interactions in Meiosis Based on the Hi-C Dataset
				3.4.1 Hi-C Reads Alignment
				3.4.2 Building Hi-C Contact Matrices and Visualization
				3.4.3 Generation of A/B Compartment Profile from Hi-C Contact Matrix
				3.4.4 Detection of Topologically Associating Domains
				3.4.5 Generation of Aggregate Hi-C Map Between Pairs of Super-Enhancers
				3.4.6 Identification of Super-Enhancer-Promoter Interactions with Virtual 4C Analysis
		4 Notes
		References
	Chapter 10: A Method for Detection of Somatic LINE-1 Insertions at the Single-Cell Level from Postmortem Human Brain
		1 Introduction
		2 Materials
			2.1 Preparation of Neural Nuclei from Human Postmortem Brain
			2.2 Whole Genome Amplification from Single Neural Nuclei (snWGA)
				2.2.1 Equipment
				2.2.2 Solutions and Reagents
			2.3 96 SNPs Genotyping for Quality Control of snWGA Products
				2.3.1 Equipment
				2.3.2 Solutions and Reagents
			2.4 Library Preparation for NECO-seq and NextSeq Run
				2.4.1 Equipment
				2.4.2 Solutions and Reagents
		3 Methods
			3.1 Preparation of Neural Nuclei from Human Postmortem Brain
			3.2 Whole Genome Amplification from Single Neural Nuclei (snWGA)
			3.3 96 SNPs Genotyping for Quality Control of snWGA Products
			3.4 Library Preparation for NECO-seq and NextSeq Run
			3.5 Detection of Novel L1Hs Insertion
		4 Notes
		References
	Chapter 11: Solubilization of Mouse Sperm Chromatin for Sequencing Analyses Using a Chaperon Protein
		1 Introduction
		2 Materials
			2.1 Preparation of Recombinant Nucleoplasmin
			2.2 Decondensation of Mouse Sperm Using Nucleoplasmin
			2.3 Chromatin Fragmentation of Nucleoplasmin-Treated Sperm
		3 Methods
			3.1 Preparation of Recombinant Nucleoplasmin
			3.2 Isolation of Mouse Sperm
			3.3 SLO Treatment of Mouse Sperm
			3.4 Nucleoplasmin Treatment of Mouse Sperm
			3.5 Chromatin Fragmentation of Nucleoplasmin-Treated Sperm
		4 Notes
		References
Part III: Manipulation of the Epigenome: Platforms for Epigenome Editing
	Chapter 12: Efficient Targeted DNA Methylation with dCas9-Coupled DNMT3A-DNMT3L Methyltransferase
		1 Introduction
		2 Materials
			2.1 gRNA Cloning Using Golden Gate Assembly
			2.2 Cell Culture
		3 Methods
			3.1 gRNA Design
			3.2 gRNA Cloning
			3.3 Transfection of Cells with EpiEditors
		4 Notes
		References
	Chapter 13: Regulation of Gene Expression Using dCas9-SunTag Platforms
		1 Introduction
		2 Materials
			2.1 Plasmids
			2.2 Preparation of Vector
			2.3 Transfection of Cells
			2.4 Gene Expression Analysis
		3 Methods
			3.1 Design and Construction of gRNA
			3.2 Transfection of Cells for Transcriptional Activation of the Target Genes
			3.3 Quantitative RT-PCR Analysis
		4 Notes
		References
	Chapter 14: Concatenated Coiled-Coil Tag for Highly Efficient, Small Molecule-Inducible Upregulation of Endogenous Mammalian G...
		1 Introduction
		2 Materials
			2.1 Construct Design and Preparation
				2.1.1 Equipment
				2.1.2 Reagents
			2.2 Mammalian Cell Culture
				2.2.1 Equipment
				2.2.2 Reagents
			2.3 Analysis of Gene Expression
				2.3.1 Equipment
				2.3.2 Reagents
		3 Methods
			3.1 Construct Design and Preparation
				3.1.1 Guide RNA Design
				3.1.2 Plasmid Preparation
			3.2 Mammalian Cell Culture
				3.2.1 Cell Cultivation
				3.2.2 Transient Transfection
				3.2.3 Small Molecule Stimulation
			3.3 Analysis of Gene Expression
				3.3.1 RNA Extraction
				3.3.2 Reverse Transcription
				3.3.3 Quantitative PCR
		4 Notes
		References
	Chapter 15: Design, Construction, and Validation of Targeted Gene Activation with TREE System in Human Cells
		1 Introduction
		2 Materials
			2.1 TREE Expression Vectors
			2.2 Cell Culture and Transfection
			2.3 Luciferase Reporter Assay
			2.4 cDNA Synthesis
			2.5 Quantification of mRNA Level on Real-Time PCR System
			2.6 Quantification of mRNA Level (ddPCR with Probe Method)
		3 Methods
			3.1 sgRNA Design and Vector Construction
			3.2 Design and Construction of Reporter Plasmid
			3.3 Lipofection
			3.4 Luciferase Assay
			3.5 cDNA Preparation
			3.6 Real-Time PCR with SYBR Green
			3.7 ddPCR with TaqMan Probes
		4 Notes
		References
Part IV: Manipulation of the Epigenome: Inducible System for Epigenome Editing
	Chapter 16: A Split CRISPR-Cpf1 Platform for Inducible Gene Activation
		1 Introduction
		2 Materials
			2.1 Media and Buffers
			2.2 Molecular Biology
			2.3 Plasmids
			2.4 Cell and Transfection Reagent
			2.5 In Vivo Experiment
			2.6 Instruments
		3 Methods
			3.1 Generation of CRISPR RNA (crRNA) Expression Vector (Fig. 2)
			3.2 Generation of crRNA Array Expression Vector
			3.3 Optogenetic Endogenous Genome Editing Experiment
			3.4 T7EI Assay for Quantifying Indel Mutation of Endogenous Genes
			3.5 Endogenous Gene Activation Experiments
			3.6 Quantitative rtPCR Analysis
			3.7 In Vivo Luciferase Reporter Activation Experiment
			3.8 In Vivo Endogenous Gene Activation Experiment
		4 Notes
		References
Part V: Manipulation of the Epigenome: Epigenetically Modified Animals
	Chapter 17: Targeted DNA Methylation in Mouse Early Embryos
		1 Introduction
		2 Materials
			2.1 Plasmid, Restriction Enzymes, and Cells
			2.2 In Vitro RNA Transcription
			2.3 Microinjection
			2.4 Live-Cell Imaging and Bisulfite Sequencing
		3 Methods
			3.1 Preparation of In Vitro Transcribed RNA
			3.2 Preparation of Fertilized Embryos and RNA Injection
			3.3 Detection of DNA Methylation and Chromatin by Live-Cell Imaging
			3.4 Quantification of Fluorescent Signals of DNA Methylation and Chromatin
			3.5 Evaluation of Induced DNA Methylation by Bisulfite Sequencing
		4 Notes
		References
	Chapter 18: Generation of Epigenetic Disease Model Mice by Targeted Demethylation of the Epigenome
		1 Introduction
		2 Materials
			2.1 Vector Construction
			2.2 Embryo Manipulation
			2.3 DNA Methylation Analysis
		3 Methods
			3.1 Vector Construction for a Single gRNA (Fig. 3a)
				3.1.1 gRNA Design
				3.1.2 Preparation of Oligonucleotides
				3.1.3 Preparation of pPlatTET-gRNA2 Vector
				3.1.4 Ligation
				3.1.5 Transformation
			3.2 Vector Construction for Multiple gRNAs (Fig. 3b)
				3.2.1 gRNA Design
				3.2.2 Preparation of Oligonucleotides
				3.2.3 Preparation of gRNA Cloning Vector BbsI
				3.2.4 Ligation
				3.2.5 Transformation
				3.2.6 Assembly of gRNA Fragments by the Golden Gate Method
				3.2.7 Preparation of Tandem gRNA Insert
				3.2.8 Preparation of pPlatTET-gRNA2 Vector
				3.2.9 Ligation and Transformation
			3.3 Microinjection of Embryos
			3.4 DNA Methylation Analysis (Adapted from)
				3.4.1 Bisulfite Modification
				3.4.2 Bisulfite PCR Amplification
				3.4.3 COBRA
				3.4.4 Bisulfite Sequencing
		4 Notes
		References
	Chapter 19: In Vivo Tissue-Specific DNA Demethylation in Mouse Liver Through a Hydrodynamic Tail Vein Injection
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Plasmids for Targeted DNA Demethylation
			2.3 Preparation of Vector
			2.4 Components Used for HTVi
			2.5 Bisulfite Sequencing (BS) Analysis
		3 Methods
			3.1 Vector Construction
				3.1.1 gRNA Design
				3.1.2 Preparation of the Double-Stranded Oligonucleotide
				3.1.3 gRNA Cloning into pPlatTET-gRNA2, All-in-One Vector
			3.2 Hydrodynamic Tail Vein Injection of the All-in-One Vector
			3.3 Bisulfite Sequencing (BS) Analysis
		4 Notes
		References
	Chapter 20: Targeted Manipulation of Histone Modification in Medaka Embryos
		1 Introduction
		2 Materials
			2.1 Preparation or Construction of dCas9-Effector Fusions
			2.2 Designing Targets and Preparation of sgRNA
			2.3 mRNA Injection into Medaka One-Cell Stage Embryo
			2.4 Crosslinking
			2.5 Sonication
			2.6 ChIP
			2.7 ChIP-qPCR
			2.8 ChIP-seq and Data Processing
		3 Methods
			3.1 Preparation or Construction of dCas9-Effector Fusions
			3.2 Designing Targets and Preparation of sgRNA
			3.3 mRNA Injection into Medaka One-Cell Stage Embryo
			3.4 Crosslinking
			3.5 Sonication
			3.6 ChIP
			3.7 ChIP-qPCR
			3.8 ChIP-seq and Data Processing
		4 Notes
		References
Index