DNA-Protein Interactions: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: A DNA Pull-Down Assay with Diversity Forms of Competitor for Detecting or Evaluating Protein-DNA Interactions
1 Introduction
2 Materials
2.1 Materials for Pull-Down Assay
2.2 Buffers and Reagents
3 Methods
3.1 Cell Lysate Extraction
3.2 Wash Dyna Magnetic Beads
3.3 Immobilize DNA
3.4 Pull-Down of DNA-Binding Proteins
3.5 Elution with LDS Sample Buffer
3.6 Results and Analysis
4 Notes
References
Chapter 2: Enhanced Yeast One-Hybrid Assays to Study Protein-DNA Interactions
1 Introduction
2 Materials
2.1 Yeast Plates
2.2 Robotic Yeast Transfer
2.3 Manual Yeast Transfer
3 Methods
3.1 Preparing Yeast Plates (See Note 2)
3.1.1 Sc-Ura-His 15 cm Round Plates
3.1.2 Sc-Trp Rectangular Plates
3.1.3 YAPD Rectangular Plates
3.1.4 Sc-Ura-Trp Rectangular Plates
3.1.5 Sc-Ura-His-Trp + 3AT + X-gal Rectangular Plates
3.2 Spotting the TF-Prey Array (See Note 6) (Fig. 2)
3.3 Preparing DNA-Bait Yeast Strain Lawns
3.4 eY1H Assay (Fig. 3)
3.4.1 Mating of DNA-Bait and TF-Prey Yeast
4 Notes
References
Chapter 3: Chromatin Immunoprecipitation on Fixed Tissues and Cell Lines
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
3 Method
3.1 Harvesting Material and Cross-Linking
3.2 Chromatin Isolation and Sonication
3.3 Immunoprecipitation
3.4 IP Washes and Cross-Link Reversal
3.5 Chromatin Purification and Quantification
4 Notes
References
Chapter 4: The ChIP-Exo Method to Identify Genomic Locations of DNA-Binding Proteins at Near Single Base-Pair Resolution
1 Introduction
2 Materials
2.1 Cell Collection and Cross-Linking
2.2 Cell Lysis and Sonication
2.3 ChIP
2.4 Enzymatic Reactions on Beads
2.5 Elution and DNA Purification
2.6 ChIP-Exo Library Preparation
3 Methods
3.1 Cell Collection and Cross-Linking
3.2 Cell Lysis and Sonication
3.3 ChIP
3.4 Enzymatic Reaction on Beads
3.5 Elution and DNA Purification
3.6 ChIP-Exo Library Preparation
4 Notes
References
Chapter 5: ChIP-Seq Protocol for In Vitro Cell Differentiation Systems
1 Introduction
2 Materials
2.1 Equipment
2.2 Software
2.3 Reagents
2.3.1 Stock Solutions (See Note 1)
2.3.2 Buffers
2.3.3 Other
3 Methods
3.1 Day 1: Preparation of Antibody-Conjugated Magnetic Beads (30 min)
3.2 Day 2: Cross-Linking and Freezing Cells (2 h)
3.3 Day 3: Sonication and Preparation of Cell Lysates (3 h)
3.4 Day 4: Immunoprecipitation (4 h)
3.5 Day 5: Cross-Link Reversal and DNA Purification (4 h)
3.6 Day 6: Library Preparation and Analysis (7 h)
4 Notes
References
Chapter 6: Profiling Accessible Chromatin and Nucleosomes in the Mammalian Genome
1 Introduction
2 Materials
2.1 Cell Permeabilization
2.2 MNase Digestion
2.3 DNA Purification
2.4 Mono- and Subnucleosome DNA Selection
3 Methods
3.1 Cell Permeabilization
3.2 MNase Digestion
3.3 DNA Purification
3.4 Mono- and Subnucleosome DNA Selection (Removal of Di-nucleosomal and Larger DNA Fragments)
3.5 Library Prep and Paired-End Sequencing
3.6 Data Processing and Analysis
4 Notes
References
Chapter 7: Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE
1 Introduction
2 Materials
2.1 Design and Generation of CRE-Targeting sgRNAs for CAPTURE
2.2 Engineer Cells for CAPTURE
2.3 Quality Control of dCas9 Enrichment at Target CREs
2.4 CAPTURE-3C-Seq
2.5 CAPTURE-Proteomics
3 Methods
3.1 Design and Generation of CRE-Targeting sgRNAs for CAPTURE
3.2 Engineer Cells for CAPTURE
3.3 Quality Control of dCas9 Enrichment at Target CREs
3.4 CAPTURE-3C-Seq
3.5 CAPTURE-Proteomics
4 Notes
References
Chapter 8: CUT & RUN to Profile Chromatin-Bound Proteins in Primary Mouse Neural Progenitor Cells
1 Introduction
2 Materials
2.1 Embryonic Brain Dissection
2.2 Brain Dissociation
2.3 FACS
2.4 CUT & RUN
2.5 Library Prep
2.6 Sequencing and Analysis
3 Methods
3.1 Mouse Embryo Dissection
3.2 NPC Dissociation and FACS
3.3 CUT & RUN
3.4 Library Preparation, Sequencing, and Analysis
4 Notes
References
Chapter 9: Easy Hi-C: A Low-Input Method for Capturing Genome Organization
1 Introduction
2 Materials
2.1 Cell Cross-Linking
2.2 Cell Lysis and HindIII Digestion
2.3 Proximity Ligation and Reverse-Linking
2.4 DpnII Digestion and Self-Circularization
2.5 Exonuclease Digestion and Re-linearization
2.6 Sequencing Library Preparation
3 Methods
3.1 Cell Preparation and Cross-Linking
3.2 Cell Lysis and HindIII Digestion
3.3 Proximity Ligation and Reverse-Linking
3.4 DpnII Digestion and Self-Circularization
3.5 Exonuclease Digestion and Re-linearization
3.6 Sequencing Library Preparation and Quantification
4 Notes
References
Chapter 10: Single Molecule Imaging of DNA-Protein Interactions Using DNA Curtains
1 Introduction
2 Materials
2.1 TIRF Microscope (See Note 1, Fig. 1)
2.2 Microfluidics for Flow Cell
2.3 Microscope Slide Nanofabrication
2.4 Flow Cell Assembly
2.5 Lipids
2.6 DNA Attachment
2.7 Lambda DNA
3 Methods
3.1 Flow Cell Assembly
3.2 Lipid Preparation
3.3 Lambda DNA Preparation
3.4 DNA Curtains Experiment
4 Notes
References
Chapter 11: Quantifying the Binding and Target-Search Kinetics of Transcriptional Regulatory Factors by Live-Cell Single-Molec...
1 Introduction
2 Materials
2.1 Cell Culture Medium
2.2 Imaging Dishes
2.3 Reagents, Peptides, and Recombinant Proteins
2.4 Recommended Cell Lines
2.5 Single-Molecule TIRF Microscopy
2.6 Software and Algorithms
3 Methods
3.1 Imaging Dish Preparation
3.2 Single-Molecule Kinetic Fraction Data Acquisition
3.3 Single-Molecule Residence Time Data Acquisition
3.4 Data Analysis
3.5 Determination of Target-Search Parameters
4 Notes
References
Chapter 12: Preparation of Recombinant Histones and Widom 601 DNA for Reconstitution of Nucleosome Core Particles
1 Introduction
2 Materials
2.1 Overexpression of Histones
2.2 Extraction from Inclusion Bodies
2.3 Histone Purification
2.4 Amplification of 32-Repeat Widom 601 DNA Plasmid
2.5 Purification of Widom 601 DNA
2.6 Equipment
3 Methods
3.1 Overexpression of Histones
3.2 Extraction of Histones from Inclusion Bodies
3.3 Purification of Histones Via Ion Exchange Chromatography
3.4 Amplification of 32-Repeat Widom 601 DNA Plasmid
3.5 Alkaline Lysis to Release 32-Repeat Widom 601 DNA Plasmid
3.6 Phenol Extraction of 32-Repeat Widom 601 DNA Plasmid
3.7 EcoRV Digestion of 32-Repeat Widom 601 DNA Plasmid
3.8 Final Purification of Widom 601 DNA
4 Notes
References
Chapter 13: Nucleosome Core Particle Reconstitution with Recombinant Histones and Widom 601 DNA
1 Introduction
2 Materials
2.1 Nucleosome Components
2.2 Buffers
2.3 Instruments and Equipment
2.4 Supplies
3 Methods
3.1 Histone Refolding of H2A/H2B Dimer and H3/H4 Tetramer
3.2 Nucleosome Core Particle Reconstitution
3.3 Nucleosome Core Particle Purification
4 Notes
References
Chapter 14: Rapid Single-Pot Assembly of Modular Chromatin Proteins for Epigenetic Engineering
1 Introduction
1.1 Synthetic Proteins That Interact with the Epigenome
1.2 Synthetic Proteins That Edit the Epigenome
1.3 Experimental Design
1.4 Approaches for Module (Donor) Construction
1.5 Destination Vectors
1.6 Testing Chromatin-Binding Fusion Proteins
1.7 Testing Epigenome Editors
2 Materials
2.1 Donor DNA Preparation
2.2 Donor DNA Preparation: Optional Donor Plasmid Cloning
2.3 Golden Gate Assembly
2.4 Colony PCR Screening
2.5 Recombinant DNA Purification and Analysis
2.6 Type IIS ``Drop-In´´ Cloning of gRNA Target Sequence for GGDestX2-Amp
2.7 Cell-Free Expression from GGDestX1-Amp Plasmids
2.8 Equipment
3 Methods
3.1 Preparation of Donor DNA Fragments
3.2 Single-Pot Assembly of Fusion Protein Plasmids
3.3 Recombinant Plasmid Screening with Colony PCR
3.4 Recombinant Plasmid Preparation and Verification with Restriction Digests
3.5 Customization of Target-Specific gRNA in GGDestX2-Amp Plasmids
3.6 Synthetic Chromatin Protein Expression In Vitro for GGDestX1-Amp Plasmids
4 Notes
References
Chapter 15: Mapping Transcription Regulation with Run-on and Sequencing Data Using the Web-Based tfTarget Gateway
1 Introduction
2 Materials
3 Methods
3.1 Setting Computing Parameters (Figs. 4 and 5)
3.2 View and Interpret the Results
4 Notes
References
Chapter 16: In Embryo Gene Reporter Assays for Evaluation of Cis-Regulatory Regions
1 Introduction
1.1 Green Fluorescent Protein (GFP) as a Reporter Gene
1.2 Luciferase as a Reporter Gene
1.3 Gene Expression Assays in Chick Embryos
2 Material
2.1 Plasmids
2.2 Electroporation and Dissection
2.3 GFP Imaging
2.4 Luciferase Assay
3 Methods
3.1 In Ovo Electroporation
3.2 Embryos Collection and Dissection
3.3 Luciferase Assay
3.4 GFP Imaging
3.5 Quantification of GFP Signal
4 Notes
References
Chapter 17: In Situ Hybridization as a Method to Examine Gene Regulatory Activity In Vivo
1 Introduction
2 Materials
2.1 Reporter Constructs and Transgenic Fly Lines
2.2 Embryo Collection and Fixation
2.3 Probe Synthesis
2.4 In Situ Hybridization
3 Methods
3.1 Cloning Reporter Constructs and Generating Transgenic Flies
3.2 Probe Synthesis
3.3 Embryo Collection and Fixation
3.4 Pre-hybridization
3.5 Hybridization
3.6 Post-hybridization and Signal Detection
4 Notes
References
Chapter 18: Investigating Tissue Regeneration Using the DUAL Control Genetic Ablation System
1 Introduction
2 Materials
2.1 Drosophila Stocks
2.2 Drosophila Stock Maintenance
2.3 Collection Cages
2.4 Immunofluorescence
2.5 Wing Size Screening
3 Methods
3.1 Preparing Drosophila Lines for Collection Cages
3.2 Cage Collections and Heat Shock
3.3 Dissection, Staining, and Mounting of Regenerating Imaginal Discs
3.4 Assay Regeneration via Adult Wing Scoring and Measurements
4 Notes
References
Chapter 19: Fishing for Developmental Regulatory Regions: Zebrafish Tissue-Specific ATAC-seq
1 Introduction
2 Materials
2.1 Equipment
2.2 Software
2.3 Reagents (See Note 1)
2.3.1 Dissections and Dissociation
Solutions (See Note 2)
Other Materials
2.3.2 FACS
Solutions
Other Materials
2.3.3 Low Input ATAC
Solutions
Other Materials
2.3.4 Enhancer Screening
Solutions/Chemicals
Other Materials
3 Methods
3.1 Dissections and Dissociations (4 h)
3.2 FACS (2 h)
3.3 Low Input ATAC-seq Protocol (4 h)
3.4 ATAC-seq Analysis (~1 Day)
3.5 Enhancer Screening and Transcription Factor Binding Site Mutagenesis
4 Notes
References
Index