Chromatin Accessibility: Methods and Protocols

Preface
Contents
Contributors
Part I: Bulk Cleavage-Based Methods
Chapter 1: Genome-Wide Mapping of Active Regulatory Elements Using ATAC-seq
1 Introduction
2 Materials
2.1 Transposition Buffers and Reagents
2.2 Library Building, Sequencing, and Quality Evaluation
2.3 General Materials and Equipment
3 Methods
3.1 Removal of Non-viable Cells (Optional)
3.2 Preparation of Nuclei
3.3 Transposition
3.4 DNA Purification
3.5 PCR Amplification and Library Generation
3.6 Library Quantification and Evaluation of Library Quality
3.7 Sequencing
4 Expected Results
5 Notes
References
Chapter 2: Mapping Nucleosome Location Using FS-Seq
1 Introduction
1.1 Basic Protocol for Preparing Sequencing Libraries from Chromatin Fragmented Using the FS Kit
2 Materials
3 Methods
3.1 Fragmentation of SV40 Chromatin Using the New England Biolabs NEXT Ultra II FS DNA Library Prep Kit
3.2 Preparation of Sequencing Libraries from FS Fragmented DNA Using the New England Biolabs NEXT Ultra II DNA Library Prep Kit
3.3 Bioinformatic Analyses
3.4 Reagents and Solutions
4 Notes
References
Chapter 3: Universal NicE-Seq: A Simple and Quick Method for Accessible Chromatin Detection in Fixed Cells
1 Introduction
2 Materials
2.1 Harvesting and Crosslinking Cells
2.2 Accessible Chromatin Labeling
2.3 Other Material and Equipment for Chromatin Labeling
2.4 Material for Quality Control
2.5 Material of the Library Construction
3 Methods
3.1 Harvesting and Crosslinking Cells
3.2 Accessible Chromatin Labeling and Decrosslinking
3.3 Labeled Genomic DNA Extraction Using Phenol Chloroform/Spin Column Method
3.3.1 Quality Control for Accessible Chromatin Labeling (Optional)
3.4 DNA Fragmentation for NGS Library
3.5 Universal NicE-seq Library Construction
3.6 PCR Cleanup Using AMPure Beads
3.7 Optional Method A: Sonication Free Labeled DNA Enrichment for NicE-seq Library Preparation Using Nicking Enzyme Digestion
3.8 Optional Method B: One Tube NicE-seq
3.8.1 Harvesting and Crosslinking Cells
3.8.2 Accessible Chromatin Labeling and Decrosslinking
3.8.3 Fragmentation of Labeled Chromatin by the Nicking Enzyme Digestion
3.8.4 DNA Pull-Down
3.8.5 End-Repair/dA Tailing
3.8.6 Adaptor Ligation
3.8.7 PCR Amplification and PCR Cleanup by AMPure Beads
3.9 Optional Method C: One Tube NicE-seq of the Human FFPE Samples from 5 to 10 μm Tissue Section
3.9.1 Removal of Paraffin from the Tissue Section Slide
3.9.2 Accessible Chromatin Labeling and Decrosslinking
3.9.3 Enrichment of Labeled Chromatin by the Nicking Enzyme Digestion
3.9.4 DNA Pull-Down
3.9.5 End-Repair/dA Tailing
3.9.6 Adaptor Ligation
3.9.7 PCR Amplification and PCR Cleanup by AMPure Beads
4 Notes
References
Chapter 4: Measuring Inaccessible Chromatin Genome-Wide Using Protect-seq
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.2 DNA Purification, Library Building, and Quality Control
2.3 Microscopy
2.4 General Material and Equipment
3 Methods
3.1 Cell Culture and Crosslinking
3.2 Nuclei Purification
3.3 Nuclease Digestion
3.4 Reverse Crosslinking and DNA Purification
3.5 NGS Library Preparation
3.6 NGS Library Quantification and Quality Control
3.7 Sequencing and Expected Results
4 Notes
References
Chapter 5: Determination of the Chromatin Openness in Bacterial Genomes
1 Introduction
2 Materials
3 Methods
3.1 Preparation of the Crosslinked Cell Lysate
3.2 Tagmentation and Preparation of the PCR Mix
3.3 Amplification of the POP-seq Libraries
3.4 Purification of the Libraries
3.5 Measuring the Library Concentration and Checking the Quality of the Libraries
3.6 Alignment of the Library Sequencing Reads to the Reference Genome
4 Notes
References
Chapter 6: Profiling Chromatin Accessibility on Replicated DNA with repli-ATAC-Seq
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.2.1 For Thymidine Chase to Study Chromatin Maturation
2.2.2 For D. melanogaster Spike-in
2.3 Buffers
3 Methods
3.1 EdU Labeling
3.2 Cell Lysis
3.3 Transposase Digestion
3.4 Click Biotinylation
3.5 Streptavidin Pulldown
3.6 Library Amplification (on Beads)
3.7 Quality Control
3.8 Sequencing and Analysis
4 Notes
References
Chapter 7: Analysis of Chromatin Interaction and Accessibility by Trac-Looping
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers
2.3 Equipment
2.4 Oligos
3 Methods
3.1 Expression and Purification of Hyperactive Tn5 and Annealing of Adapters
3.2 Cell Fixation
3.3 Assemble the Tn5 Complex and DNA Transposition Reaction
3.4 Reverse Cross-Linking and Purify Genomic DNA
3.5 Repair DNA Gaps Between the Bivalent Adapter and Genomic DNA
3.6 DNA Restriction Enzyme Digestion and Enrichment via the Biotinylated Bivalent Adapter
3.7 Self-Circularization of Genomic DNA Fragments in a Large Volume
3.8 RCA Reaction in a Small Volume
3.9 Library Indexing and Amplification
4 Notes
References
Part II: Methods for Measuring the Absolute Levels of Occupancy/Accessibility
Chapter 8: Single-Molecule Mapping of Chromatin Accessibility Using NOMe-seq/dSMF
1 Introduction
2 Materials
2.1 Methylation Buffers and Reagents
2.2 Library Building, Sequencing, and Quality Evaluation
2.3 General Materials and Equipment
2.4 Software Packages
3 Methods
3.1 Preparation of Nuclei
3.2 Methylation Treatment
3.3 DNA Purification
3.4 Library Preparation-Whole-Genome SMF
3.5 Library Preparation-Probe-Hybridization Enrichment
3.6 Library Preparation-Amplicon-Targeted SMF
3.7 Library Quantification and Evaluation of Library Quality
3.8 Sequencing
3.9 Computational Analysis
3.9.1 Adapter Trimming
3.9.2 Read Mapping and Alignment Filtering
3.9.3 Methylation Conversion Assessment
3.9.4 Methylation Calling
3.9.5 Bulk Accessibility or Methylation Profile Generation
3.9.6 Metaprofile Evaluation
3.9.7 Generating Single-Molecule Maps
4 Expected Results
5 Notes
References
Chapter 9: ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities
1 Introduction
2 Materials
2.1 Cells and Buffers for Preparation of S. cerevisiae Chromatin
2.2 Cells and Buffers for Preparation of S. cerevisiae and S. pombe Genomic DNA (gDNA)
2.3 Buffers and Enzymes for Digestion of Chromatin and S. cerevisiae/S. pombe gDNA with Restriction Enzymes and DNA Purificati...
2.4 DNA Shearing and Purification After RE Digestion and Preparation of DNA Sequencing Libraries
3 Methods
3.1 Preparation of S. cerevisiae Chromatin (``Nuclei,´´ See Note 2)
3.2 Preparation of S. cerevisiae and S. pombe gDNA (See Note 10)
3.3 Optional: Restriction Enzyme Digest of S. pombe gDNA
3.4 Chromatin Digest with Restriction Enzymes and DNA Purification
3.5 Optional: Second RE Digest for Cut-All Cut Method
3.6 DNA Shearing and Purification
3.7 Sequencing Library Preparation
3.8 Calibration Curve
3.9 Bioinformatics Analysis
3.9.1 Sample Naming Rules
3.9.2 How to Use Other REs
3.9.3 How to Use Other Genomes
3.9.4 How to Fit Uncut Correction Factors
4 Notes
4.1 Bioinformatic Notes
4.1.1 Map/Filter Reads
4.1.2 Count Cut and Uncut Fragments
4.1.3 Determine Cut Site Positions with RE Motif
4.1.4 Remove RE Sites with Close Neighbor RE Sites
4.1.5 Collect Cut and Uncut Counts Within Window Near Cut Sites to Correct for Resection
4.1.6 Occupancy Estimation by Cut-All Cut Method with Background Correction and Normalization
4.1.7 Occupancy Estimation by Cut-Uncut Method with Background Correction
References
Part III: Methods for Profiling Chromatin Accessibility at the Single-Cell Level
Chapter 10: Single-Cell Joint Profiling of Open Chromatin and Transcriptome by Paired-Seq
1 Introduction
2 Materials
2.1 Reagents Preparation
2.2 Nuclei Isolation
2.3 Chromatin Tagmentation
2.4 Reverse Transcription
2.5 Adding DNA Barcodes
2.6 Library Pre-amplification
2.7 Library Splitting
2.8 Library Amplification
2.9 Sequencing and Data Preprocessing
3 Methods
3.1 Reagents Preparation
3.2 Nuclei Isolation
3.3 Chromatin Tagmentation
3.4 Reverse Transcription
3.5 Adding DNA Barcodes
3.6 Library Pre-amplification
3.7 Library Splitting
3.8 Library Amplification
3.9 Sequencing and Data Preprocessing
4 Notes
References
Chapter 11: Simultaneous Single-Cell Profiling of the Transcriptome and Accessible Chromatin Using SHARE-seq
1 Introduction
2 Materials
2.1 DNA Oligos and Primers
2.2 General Reagents
2.3 General Equipment
2.4 Buffers and Reagents
2.5 Software Packages
3 Methods
3.1 Determining the Optimal Cell Number
3.2 Annealing of Oligo Plates
3.3 Anneal Adapter Oligos
3.4 Transposome Assembly
3.5 Tissue Dissociation
3.6 Fixation of Cells in Culture and of Dissociated Nuclei from Tissue
3.7 ATAC Reaction
3.8 Reverse Transcription
3.9 Hybridization-Ligation and Pool-Split
3.10 Reverse Crosslinking
3.11 Pulldown
3.12 ATAC Library Preparation
3.13 RNA Library Preparation Step 1. Template Switching
3.14 RNA Library Preparation Step 2. Amplification of cDNA
3.15 RNA Library Preparation Step 3. Tagmentation
3.16 RNA Library Preparation Step 4. Final Amplification
3.17 Library Quantification and Evaluation of Library Quality
3.18 Sequencing
4 Computational Processing
4.1 RNA
4.2 ATAC
5 Expected Results
5.1 Sequencing Libraries
5.2 Species Mixing Experiments
5.3 ATAC Post-sequencing Quality Evaluation
5.4 RNA Post-sequencing Quality Evaluation
5.5 Dimensionality Reduction and Cell Type/Cluster Identification
6 Notes
References
Chapter 12: Simultaneous Measurement of DNA Methylation and Nucleosome Occupancy in Single Cells Using scNOMe-Seq
1 Introduction
2 Materials
2.1 Reagents/Consumables
2.1.1 Nuclei Isolation and GpC Methyltransferase Treatment
2.1.2 Fluorescence-Activated Cell Sorting
2.1.3 Bisulfite Conversion
2.1.4 Random-Primed DNA Synthesis
2.1.5 Inactivation of Free Primers and dNTPs
2.1.6 Sample Clean-Up
2.1.7 Adaptase Reaction
2.1.8 Library Amplification
2.1.9 Library Clean-Up
2.1.10 Primers and Barcodes
2.2 Equipment
2.2.1 Assay
2.2.2 Sequencing
3 Methods
3.1 Assay
3.1.1 Nuclei Isolation and GpC Methyltransferase Treatment
3.1.2 Fluorescence-Activated Cell Sorting
3.1.3 Bisulfite Conversion
3.1.4 Random-Primed DNA Synthesis
3.1.5 Inactivation of Free Primers and dNTPs
3.1.6 Sample Clean-Up
3.1.7 Adaptase Reaction
3.1.8 Library Amplification
3.1.9 Library Clean-Up
3.2 Sequencing
3.3 Analysis
4 Notes
References
Chapter 13: Massively Parallel Profiling of Accessible Chromatin and Proteins with ASAP-Seq
1 Introduction
2 Materials
2.1 Cell Processing, Staining, Fixation, and Lysis
2.2 ASAP-Seq Library Preparation
2.3 Quality Control and Sequencing
2.4 Software and References Needed for Computational Analysis
3 Methods
3.1 Cell Preparation, Fixation, and Permeabilization
3.1.1 Cell Staining
3.1.2 Cell Fixation and Permeabilization
3.1.3 Intracellular Staining
3.2 Transposition and Barcoding
3.3 Library QC, Pooling and Sequencing
3.3.1 Library QC
3.3.2 Pooling and Sequencing
Box 1: ASAP-Seq Tag Libraries Structure and Sequencing Schemes
3.4 Demultiplex Sequencing Data
3.5 Process Sequencing Data
Box 2: Example of Quality Metrics from Running Steps in Subheading 3.5. Key Metrics Are Indicated After the Associated Computa...
3.6 Perform Multimodal Analysis
4 Notes
References
Chapter 14: Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq
1 Introduction
2 Materials
2.1 Cell Processing, Fixation, and Lysis
2.2 mtscATAC-Seq Library Preparation
2.3 Quality Control and Sequencing
2.4 Computational Resources
3 Methods
3.1 Cell Processing, Fixation, and Lysis
3.2 mtscATAC-Seq Library Preparation
3.3 Quality Control and Sequencing
3.4 Computational Processing and Analyses
4 Notes
References
Part IV: Imaging Methods for Visualization of Accessible DNA
Chapter 15: ATAC-See: A Tn5 Transposase-Mediated Assay for Detection of Chromatin Accessibility with Imaging
1 Introduction
2 Materials
2.1 Hyperactive Tn5 Production
2.2 Tn5 Transposase Assembly
2.3 Tn5 Tagmentation
2.4 Immunostaining of Mitochondria and Nuclear Lamina
2.5 Cell Culture
2.6 Equipment
3 Methods
3.1 Hyperactive Tn5 Production
3.2 Tn5 Transposome Assembly
3.3 Slide Preparation and Fixation
3.4 ATAC-see
3.5 Immunostaining After ATAC-see
4 Notes
References
Chapter 16: NicE-viewSeq: An Integrative Visualization and Genomics Method to Detect Accessible Chromatin in Fixed Cells
1 Introduction
2 Materials
2.1 Crosslinking Cells on Micro Cover Glass
2.2 Accessible Chromatin Labeling
2.3 Mounting Slides and Open Chromatin Index Using Zeiss LSM 880 Confocal Microscope
2.4 NicE-viewSeq Library Construction
3 Methods
3.1 Crosslinking Cells on Micro Cover Glass
3.2 Accessible Chromatin Labeling
3.3 Mounting Slides and Open Chromatin Index Using Zeiss LSM 880 Confocal Microscope
3.4 NicE-viewSeq DNA Preparation
3.5 NicE-viewSeq DNA Pull-Down on Beads
3.6 NicE-viewSeq NGS Library Preparation
3.7 NicE-viewSeq NGS Library Amplification
3.8 NicE-viewSeq NGS Library Purification Using NEBNext Sample Purification Beads
4 Notes
References
Part V: Computational Analysis of Chromatin Accessibility Datasets
Chapter 17: ATAC-seq Data Processing
1 Introduction
2 Materials
3 Methods
3.1 Adaptor Detection and Read Trimming
3.2 Read Alignment and Post-alignment Filtering
3.3 Peak Calling
3.4 Identifying Replicate-Consistent Peaks
3.5 Generating Signal Tracks
3.6 ATAC-seq Quality Control Evaluation
4 Notes
References
Chapter 18: Deep Learning on Chromatin Accessibility
1 Introduction
2 Materials
3 Methods
3.1 Data Processing and Data Loading
3.2 Train a Model
3.3 Evaluation
3.4 Inference
4 Notes
References
Index