Chromatin: Methods and Protocols

Preface
Contents
Contributors
Part I: DNA Methylation
	Chapter 1: Generating Sequencing-Based DNA Methylation Maps from Low DNA Input Samples
		1 Introduction
		2 Materials
			2.1 Experimental Workflow
			2.2 Data Analysis
		3 Methods
			3.1 DNA Purification
			3.2 Restriction Endonuclease Digestion (MspI)
			3.3 End Repair
			3.4 Adenylation of 3′ Ends
			3.5 Methylated Adapter Ligation
			3.6 Bisulfite Conversion
			3.7 Determine the Optimal Amplification Cycles by Semiquantitative Amplification
			3.8 Large-Scale PCR Amplification
			3.9 Size Selection with Beads
			3.10 Quantitative and Qualitative Assessment of the Library
			3.11 Cluster Generation for Multiplexed RRBS
			3.12 Sequencing and Methylation Calls
			3.13 Assessing Data Quality and Alignment
			3.14 Tools for Detecting Differential Methylation from RRBS Data
		4 Notes
		References
	Chapter 2: Data Analysis of DNA Methylation Epigenome-Wide Association Studies (EWAS): A Guide to the Principles of Best Pract...
		1 Introduction
		2 EWAS Design Considerations
			2.1 Study Type
			2.2 Phenotype/Trait Selection
			2.3 Sample Type and Quality
			2.4 Cellular Heterogeneity
			2.5 Sample Size and Independent Validation
		3 Data Analysis
			3.1 Data Preprocessing
				3.1.1 Normalization
				3.1.2 QC and Data Filtering
			3.2 EWAS Analysis Using ChAMP; the Chip Analysis Methylation Pipeline
				3.2.1 Data Loading
				3.2.2 Data Filtering and QC
				3.2.3 Data Normalization
				3.2.4 Differential Methylation Probes
			3.3 Independence of Association/Adjusting for Confounding Factors
			3.4 Singular Value Decomposition (SVD) Analysis and Adjusting for Confounding Factors
		4 Interpreting the Data Output
			4.1 Data Visualization
			4.2 Interpreting Top Hit Associations
			4.3 Identification of Methylation Quantitative Trait Loci (meQTLs)
		References
	Chapter 3: Next-Generation Bisulfite Sequencing for Targeted DNA Methylation Analysis
		1 Introduction
		2 Materials
			2.1 Bisulfite-Specific Primer Design
			2.2 Genomic DNA Purification and Bisulfite Conversion
			2.3 Bisulfite-Specific PCR Amplification.
			2.4 Agarose Gel Electrophoresis
			2.5 PCR Clean-Up
			2.6 PCR Product Quantitation
			2.7 Sequencing Library Quality Assessment
			2.8 High-Throughput Sequencing
			2.9 Downstream Analysis of High-Throughput Sequencing  Data
		3 Methods
			3.1 Bisulfite-Specific PCR Primer Design
			3.2 DNA Purification and Bisulfite Conversion
			3.3 First Round Touchdown  PCR
			3.4 Bead Clean-Up of PCR Products (See Note 9)
			3.5 Qubit Concentration and Dilution
			3.6 Second Round PCR
			3.7 Library Preparation and Bead Clean-Up
			3.8 Library Quality Check
			3.9 MiSeq Sequencing
			3.10 Sequencing Analysis
		4 Notes
		References
	Chapter 4: Editing of DNA Methylation Patterns Using CRISPR-Based Tools
		1 Introduction
		2 Materials
			2.1 gRNA Design
			2.2 gRNA and Other Plasmid Construct Preparation
			2.3 Cell Culture and Transfection
			2.4 FACS
		3 Methods
			3.1 gRNA Design and Preparation
			3.2 CRISPR-dCas9-SunTag and Effector Construct Preparation
			3.3 CRISPR-Methylation Editing System Transfection
			3.4 FACS for Edited Cell Collection
		4 Notes
		References
	Chapter 5: Nanopore Sequencing and Data Analysis for Base-Resolution Genome-Wide 5-Methylcytosine Profiling
		1 Introduction
		2 Materials
			2.1 Zebrafish Embryos
			2.2 Phenol-Chloroform Genomic DNA Extraction Followed by Ethanol Precipitation
			2.3 Genomic DNA Quality Control
				2.3.1 Size Selection (Optional)
			2.4 Library Preparation and Sequencing
			2.5 Base-Calling, Read Alignment and Analysis
		3 Methods
			3.1 Genomic DNA Extraction
				3.1.1 Phenol-Chloroform Genomic DNA Extraction Followed by Ethanol Precipitation
				3.1.2 Alternative Purification of Genomic  DNA
			3.2 Quality Control
			3.3 Library Preparation and Sequencing
			3.4 Read Alignment and Methylation Calling
				3.4.1 Data Download
				3.4.2 Nanopore Base-Calling and Methylation Profiling
				3.4.3 WGBS Data Assembly and Methylation Calling
			3.5 Example Downstream Analyses
				3.5.1 Bedgraph File Structure
				3.5.2 CpG Count
				3.5.3 Genomic Tracks Coverage
				3.5.4 Methylation Analysis
		4 Notes
		References
Part II: Protein-DNA Interactions
	Chapter 6: Chromatin Immunoprecipitation Sequencing (ChIP-seq) Protocol for Small Amounts of Frozen Biobanked Cardiac Tissue
		1 Introduction
		2 Materials
			2.1 Chemicals and Reagents
			2.2 Working Buffers
			2.3 Equipment
		3 Methods
			3.1 Preparing Tissue and Cross-Linking the Chromatin
			3.2 Lysing the Cells
			3.3 Shearing the Chromatin
			3.4 Diluting the Chromatin
			3.5 Coupling the Antibody to Dynabeads
			3.6 Binding Chromatin to the Beads
			3.7 Washing the Bound Chromatin
			3.8 Reversing the Cross-Linking
			3.9 Purifying the  DNA
			3.10 End-Pair and Adenylation of the Purified DNA Fragments
			3.11 Adaptor Ligation
			3.12 Cleanup
			3.13 PCR Amplification
		4 Notes
		References
	Chapter 7: A Robust Protocol for Investigating the Cohesin Complex by ChIP-Sequencing
		1 Introduction
		2 Materials and Equipment
			2.1 Cross-Linking and Cell Harvesting
			2.2 Sonication
			2.3 Chromatin Immunoprecipitation and DNA Cleanup
		3 Methods
			3.1 Cross-Linking and Cell Harvesting
			3.2 Sonication
			3.3 Immunoprecipitation
			3.4 Elution and Reversal of Cross-Links
			3.5 DNA Cleanup
			3.6 Analysis and Preparation for Sequencing
		4 Notes
		References
	Chapter 8: Epi-Decoder: Decoding the Local Proteome of a Genomic Locus by Massive Parallel Chromatin Immunoprecipitation Combi...
		1 Introduction
		2 Materials
			2.1 Yeast Strains, Plasmids and Oligos
				2.1.1 Yeast Strains
				2.1.2 Plasmids
				2.1.3 Oligos
			2.2 Yeast Drugs and Media
			2.3 Buffers
			2.4 Reagents
			2.5 Equipment
		3 Methods
			3.1 Design and Construction of the Barcoder Library
				3.1.1 Preparation of the Yeast Strain for the Barcoder Library
				3.1.2 Generating the Cas9/gRNA Vector
				3.1.3 Preparation of the Barcode-Containing Repair Template
				3.1.4 Construction of the Barcoder Library
				3.1.5 Decoding the Barcode Sequences and Locations in the Barcoder Library
			3.2 Generating the Epi-Decoder Library
			3.3 Epi-Decoder: TAG-ChIP-Barcode-Seq
				3.3.1 Cross-Linking and Pellet Collection
				3.3.2 Chromatin Preparation
				3.3.3 Coupling Epoxy-Activated Dynabeads (IgG Dynabeads)
				3.3.4 IP, Wash and DNA Clean-Up
				3.3.5 Library Preparation and Barcode Counting
		4 Notes
		References
	Chapter 9: A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based A...
		1 Introduction
		2 Materials
			2.1 Reagents Needed for Cell Preparation
			2.2 Microscope Set-Up
			2.3 Localization and Tracking
			2.4 Analysis Using Spot-On
		3 Methods
			3.1 Cell Preparation
			3.2 Imaging
			3.3 Trajectory Generation
			3.4 Trajectory Analysis with Spot-On
		4 Notes
		References
	Chapter 10: Characterization of Mammalian Regulatory Complexes at Single-Locus Resolution Using TINC
		1 Introduction
		2 Materials
			2.1 Generation of TALE-Expressing Cell Lines
			2.2 Crosslinking of the Cells
			2.3 Isolation of Nuclei and Chromatin
			2.4 Shearing of Isolated Chromatin
			2.5 TALE-Chromatin Immunoprecipitation
			2.6 In-Gel Tryptic Digest
		3 Methods
			3.1 Generation of TALE-Expressing Cell Lines
			3.2 Crosslinking of the Cells
			3.3 Isolation of Nuclei and Chromatin
			3.4 Shearing of Isolated Chromatin
			3.5 TALE-Chromatin Immunoprecipitation
			3.6 In-Gel Tryptic Digest (See Note 17)
			3.7 Mass Spectrometry
			3.8 Mass Spectrometric Data Analysis
		4 Notes
		References
	Chapter 11: Profiling Protein-DNA Interactions Cell-Type-Specifically with Targeted DamID
		1 Introduction
		2 Materials
			2.1 Tissue Preparation
			2.2 DNA Extraction
			2.3 DpnI Digestion
			2.4 Adaptor Ligation
			2.5 DpnII Digestion
			2.6 PCR Amplification
			2.7 Sonication and Removal of DamID Adaptors
			2.8 Sequencing Library Preparation
				2.8.1 DNA Cleanup
				2.8.2 Concentration Adjustment
				2.8.3 End Repair
				2.8.4 Adenylation of 3′ Ends and Adaptor Ligation
				2.8.5 DNA Cleanup
				2.8.6 DNA Fragment Enrichment
				2.8.7 DNA Cleanup
				2.8.8 Library Quality Control
		3 Methods
			3.1 Tissue Preparation
			3.2 DNA Extraction
			3.3 DpnI Digestion
			3.4 Adaptor Ligation
			3.5 DpnII Digestion
			3.6 PCR Amplification
			3.7 Sonication and Removal of DamID Adaptors
			3.8 Sequencing Library Preparation
				3.8.1 DNA Cleanup
				3.8.2 Concentration Adjustment
				3.8.3 End Repair
				3.8.4 Adenylation of 3′ Ends
				3.8.5 Adaptor Ligation
				3.8.6 DNA Cleanup
				3.8.7 DNA Fragment Enrichment
				3.8.8 DNA Cleanup
				3.8.9 Library Quality Control
			3.9 Processing of Sequencing Data
		4 Notes
		References
	Chapter 12: Genome-Wide Mapping and Microscopy Visualization of Protein-DNA Interactions by pA-DamID
		1 Introduction
		2 Materials
			2.1 pA-DamID Localization and Activation
			2.2 Enrichment of m6A Labeled DNA and Preparation of Illumina Sequencing Library
			2.3 Visualization of m6A Labeled  DNA
			2.4 Equipment and Labware
		3 Methods
			3.1 pA-Dam Activity Testing
			3.2 pA-Dam Localization and Activation
			3.3 Enrichment of m6A Methylated DNA and Preparation of Illumina Sequencing Library
			3.4 Visualization of m6A Labeled  DNA
		4 Notes
		References
	Chapter 13: The dCypher Approach to Interrogate Chromatin Reader Activity Against Posttranslational Modification-Defined Histo...
		1 Introduction
		2 Materials
			2.1 General Reagents
			2.2 Buffers
			2.3 Equipment
		3 Methods
			3.1 Query Titration, Target Ranking
			3.2 2D [Query vs. Salt] Titrations
			3.3 2D [Query vs. Salmon Sperm DNA (SalDNA)] Titration
			3.4 Preparation of Discovery Screen Plate(s)
			3.5 Discovery Screen
		4 Notes
		References
Part III: Chromatin Accessibility
	Chapter 14: High-Resolution ATAC-Seq Analysis of Frozen Clinical Tissues
		1 Introduction
		2 Materials
			2.1 Tissue Freezing
			2.2 Tissue Sectioning and Histological Analysis
			2.3 Tissue Homogenization
			2.4 Nuclei Counting
			2.5 ATAC-Seq Transposition (Experiment in Bulk)
			2.6 ATAC-Seq Amplification
		3 Methods
			3.1 Tissue Freezing
				3.1.1 Snap Freezing
				3.1.2 OCT Embedding and Freezing
			3.2 Tissue Sectioning
			3.3 Tissue Homogenization and Cell Lysis
			3.4 Bulk ATAC-Seq
			3.5 DNA Amplification
		4 Notes
		References
	Chapter 15: Single-Molecule Multikilobase-Scale Profiling of Chromatin Accessibility Using m6A-SMAC-Seq and m6A-CpG-GpC-SMAC-S...
		1 Introduction
		2 Materials
			2.1 SMAC-Seq Buffers and Reagents
			2.2 DNA Isolation and Size Selection
			2.3 Nanopore Sequencing Flow Cells and Reagents
			2.4 General Materials and Equipment
			2.5 Computational Resources
			2.6 Genomic Sequence and Annotation Files
			2.7 Software Packages
		3 Methods
			3.1 Nuclei Isolation (Budding Yeast)
			3.2 Enzymatic Treatment of Chromatin for m6A-GpC-CpGSMAC-Seq (Budding Yeast)
			3.3 Nuclei Isolation for Human, Drosophila, and Other Cells Without Cell Walls
			3.4 Enzymatic Treatment of Chromatin for m6A-SMAC-Seq (Human Cells)
			3.5 Enzymatic Treatment of Chromatin for m6A-GpC-CpGSMAC-Seq (Drosophila Cells)
			3.6 HMW DNA Isolation
			3.7 DNA Size Selection
			3.8 Nanopore Library Construction and Sequencing
			3.9 Computational Analysis
				3.9.1 Read Mapping and Modification Calling
				3.9.2 Tombo Extraction
				3.9.3 Read Mapping and Modification Calling Using Megalodon
				3.9.4 Megalodon Per-Read Modification Extraction
				3.9.5 Merging and Indexing
				3.9.6 Calculate Mapping Statistics
				3.9.7 Create Coverage  File
				3.9.8 Bayesian Integration
				3.9.9 Filtering Fully Methylated Reads
				3.9.10 Create Genome Browser Tracks
				3.9.11 Making Metaplots Around a Position
				3.9.12 Making Single Molecule Plots
				3.9.13 Calculating NMI Matrices
		4 Notes
		References
Part IV: Genome Structure and Organization
	Chapter 16: Circular Chromosome Conformation Capture Sequencing (4C-Seq) in Primary Adherent Cells
		1 Introduction
		2 Materials
			2.1 Solutions, Reagents, and Consumables
			2.2 Equipment
		3 Methods
			3.1 4C-seq Design
			3.2 Crosslinking of Cells
			3.3 Lysis and Digestion
			3.4 Ligation
			3.5 Reverse-Crosslinking, RNase A Treatment, and DNA Extraction
			3.6 Second Digestion
			3.7 Second Ligation and Clean-up of  DNA
			3.8 4C-seq PCR
			3.9 Library Preparation
			3.10 Deep-Sequencing
			3.11 Data Processing and Analysis Options
		4 Notes
		References
	Chapter 17: Mammalian Micro-C-XL
		1 Introduction
		2 Materials
			2.1 Prepare Cross-Linked Chromatin from Cell Culture
			2.2 MNase Titration and MNase Digestion of Chromatin
			2.3 MNase Digestion of Chromatin (See Note 1)
			2.4 Dinucleosomal DNA Purification
			2.5 Streptavidin Pull-down and on-Bead Library Preparation
			2.6 Library Amplification
		3 Methods
			3.1 Prepare Cross-Linked Chromatin from Cell Culture
			3.2 MNase Titration (See Note 3)
			3.3 Preparative MNase Digestion of Chromatin
			3.4 DNA and Processing and Proximity Ligation
			3.5 Dinucleosomal DNA Purification
			3.6 Streptavidin Pull-down and On-bead Library Preparation
			3.7 Library Amplification
		4 Notes
		References
	Chapter 18: In Situ HiC
		1 Introduction
		2 Materials
		3 Method
			3.1 Cross-Linking
			3.2 Lysis and Restriction Digestion
			3.3 Tagging of DNA Ends, Proximity Ligation, and Cross-Link Reversal
			3.4 DNA Shearing and Size Selection
			3.5 Biotin Pull-down and Preparation for Illumina Sequencing
			3.6 Final Amplification and Purification
		4 Notes
		References
	Chapter 19: LncRNA-Chromatin Pull-Down Using Biotin-Conjugated DNA Probes
		1 Introduction
		2 Materials
			2.1 Buffers and Reagents
			2.2 Cell Culture
			2.3 Other Equipment
		3 Methods
			3.1 Harvesting Mammalian Cells
			3.2 Crosslinking Using 1% Glutaraldehyde to Conserve Chromatin and RNA Interactions
			3.3 Cell Lysis and Sonication to Shear Chromatin
			3.4 Chromatin Pull-Down Using Biotinylated DNA Probes
			3.5 RNA Isolation for Quantification Using qRT-PCR to Check RNA Enrichment
			3.6 DNA Isolation from Pull-Down Samples from Beads for Downstream Analysis
			3.7 RNA Pull-Down Validation Using qRT-PCR and DNA qPCR Analysis
		4 Notes
		References
	Chapter 20: Superresolution Microscopy for Visualization of Physical Contacts Between Chromosomes at Nanoscale Resolution
		1 Introduction
		2 Materials
			2.1 Preparation of Mitotic Chromosome Spreads
			2.2 Fluorescent In Situ Hybridization
			2.3 Washes and Mounting
			2.4 Immunofluorescence Labeling
			2.5 Microcopy Hardware and Software Package
		3 Methods
			3.1 Preparation of Mitotic Chromosome Spreads
			3.2 Fluorescent In Situ Hybridization (FISH)
			3.3 Posthybridization Washes and Mounting or Preparation for Immunostaining
			3.4 Immunofluorescence Labeling
			3.5 Super-Resolution Microscopy (SIM and STED)
				3.5.1 SIM Image Acquisition and Validation
				3.5.2 STED Image Acquisition and Processing
		4 Notes
		References
Index