Spatial Genome Organization: Methods and Protocols (Methods in Molecular Biology, 2532)

Description

This detailed volume explores a variety of cutting-edge techniques used to interrogate spatial genome organization. Beginning with a section covering the vital chromosome conformation capture (3C) technique, this collection continues with chapters on targeted Hi-C approaches, sequencing-based approaches to assess nuclear environment, as well as single-cell technologies to better characterize the heterogeneity and dynamics of nuclear architectures and approaches to visualize them by microscopy. Finally, in order to be able to ask functional questions about the role of spatial chromatin organization in genomic control, the last section provides methods for acute manipulations of chromatin architecture. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Spatial Genome Organization: Methods and Protocols is an ideal resource for researchers searching for the best techniques to address their own specific research questions. Preface Contents Contributors Part I: Sailing the `3Cs´: Chromosome Conformation Capture and Its Variants Chapter 1: Quantitative Chromosome Conformation Capture (3C-qPCR) 1 Introduction 2 Materials 3 Methods 3.1 Cell Nucleus Preparation 3.2 Formaldehyde Cross-Linking 3.3 Restriction Digestion 3.4 Determination of Digestion Efficiency 3.5 Ligation 3.6 DNA Purification 3.7 Complementary Digestion 3.8 Performing Loading Adjustments 3.9 Assessment of Sample Purity (Optional) 3.10 Real-Time PCR Quantifications of Ligation Products 3.11 PCR Control Template Used for Primer Efficiency Control 3.12 3C-qPCR Data Normalization-Primer Efficiency and Loading Controls 3.13 3C-qPCR Data Normalization to Noise Band 4 Notes References Chapter 2: Detection of Allele-Specific 3D Chromatin Interactions Using High-Resolution In-Nucleus 4C-seq 1 Introduction 2 Materials 3 Methods 3.1 Design of Allele-Specific 4C Primers 3.2 Cell Cross-Linking 3.3 Cell Lysis and Nucleus Permeabilization 3.4 First Digestion 3.5 DNA Digestion Efficiency 3.6 Restriction Enzyme Inactivation and Ligation 3.7 DNA Purification 3.8 Secondary Digestion and Ligation 3.9 DNA Purification 3.10 4C-seq Test PCR 3.11 Preparation of 4C-seq Illumina Sequencing Material 3.12 Data Analysis 4 Notes References Chapter 3: Tough Tissue Hi-C 1 Introduction 2 Materials 3 Methods 3.1 Tissue Collection, Formaldehyde Cross-Linking, and Nuclei Isolation 3.2 Digestion 3.3 Ligation and DNA Purification 3.4 Library Preparation (See Note 32) 4 Notes References Chapter 4: Mapping Mammalian 3D Genomes by Micro-C 1 Introduction 2 Materials 2.1 Chromatin Cross-Linking 2.2 Micrococcal Nuclease Digestion and Titration 2.3 DNA Fragment End Repair 2.4 Proximity Ligation, Removal of Unligated Ends, and Reverse Cross-Linking 2.5 Dinucleosomal DNA Purification 2.6 Library Preparation 3 Methods 3.1 Preparation of Cross-Linked Chromatin from Cell Culture 3.2 Micrococcal Nuclease Titration (See Note 14) 3.3 Digestion of Cross-Linked Chromatin with Micrococcal Nuclease 3.4 DNA Fragment End Repair 3.5 Proximity Ligation, Removal of Unligated Ends, and Reverse Cross-Linking 3.6 Dinucleosomal DNA Purification 3.7 Library Preparation 3.8 Data Analysis 3.8.1 Preprocessing Micro-C Contact Pairs 3.8.2 Convert Tab-Delimited Files to HDF5 (Cool) Format and Normalization 4 Notes References Part II: Targeted Hi-C Approaches Chapter 5: Targeted Chromosome Conformation Capture (HiCap) 1 Introduction 2 Materials 3 Methods 3.1 Probe Design 3.1.1 Preparing Target Files 3.1.2 Completing Probe Design 3.2 Cell Fixation 3.2.1 Adherent Cells (Grown on 100 Mm Cell Culture Dishes) 3.2.2 Suspension Cells 3.3 Hi-C 3.4 Removal of Biotin Moieties from Ends 3.5 Sample Shearing 3.6 Capture of Biotinylated Fragments 3.7 Sequencing Library Preparation 3.8 Library Amplification 3.9 Target Enrichment 4 Notes References Chapter 6: Assessment of Multiway Interactions with Tri-C 1 Introduction 2 Materials 2.1 3C Library Preparation 2.2 Sonication and Addition of Sequencing Adaptors 2.3 Capture Enrichment 3 Methods 3.1 3C Library Preparation 3.1.1 Fixation 3.1.2 Digestion (See Note 4) 3.1.3 Ligation and Decrosslinking 3.1.4 DNA Extraction 3.1.5 Quality Control of 3C Library 3.2 Sonication and Addition of Sequencing Adaptors 3.3 Capture Enrichment 3.3.1 Preparation of Capture Oligonucleotides (See Notes 11 and 12) 3.3.2 Hybridization Reaction 3.3.3 Binding to Streptavidin Beads 3.3.4 Amplification of Captured DNA 3.3.5 Double Capture 3.3.6 Sequencing 3.3.7 Analysis 4 Notes References Chapter 7: Assessing Specific Networks of Chromatin Interactions with HiChIP 1 Introduction 2 Materials 2.1 Hi-C (Cross-Linking, Digestion, and Ligation) 2.2 Sonication and Chromatin Immunoprecipitation 2.3 Biotin Pull-Down with Streptavidin Beads 2.4 Library Preparation for Sequencing 3 Methods 3.1 Cross-Linking, Digestion, and Ligation 3.2 Sonication and Chromatin Immunoprecipitation 3.3 Biotin Pull-Down 3.4 Library Preparation 3.5 Analysis 3.5.1 Use of HiC-Bench 3.5.2 Alignment, Filtering and Extraction of ``Usable´´ Reads 3.5.3 HiChIP Stats and QCs 3.5.4 Generation of Interaction Matrices 3.5.5 File Transformation for Visualization in JuiceBox 3.5.6 Identification of Significant Interactions 3.5.7 Categorization of HiChIP Loops 3.5.8 Comparison of Different Loop Calling Methods 3.5.9 Identification of Differential Interactions Between MEFs and ESCs 4 Notes References Part III: Sequencing-Based Approaches to Assess Nuclear Environment Chapter 8: Measuring Cytological Proximity of Chromosomal Loci to Defined Nuclear Compartments with TSA-seq 1 Introduction 1.1 Overview 1.2 General Guide to Designing a Tailored TSA-seq Protocol 1.3 Nuclear Compartment Target and Antibody Selection 1.4 Testing TSA Staining Conditions 1.5 Scaling Up TSA Staining for the Larger Cell Numbers Required for TSA-seq 1.6 Harvesting and Lysing Cells, DNA Purification and Sonication, and Assaying DNA Biotinylation 1.7 Pulldown of Biotinylated DNA 1.8 DNA Library Preparation, Sequencing and Data Processing 1.9 Summary 2 Materials 2.1 TSA Cell Labeling 2.2 Immunostaining to Check Tyramide-Biotin Labeling 2.3 Genomic DNA Extraction and Sonication 2.4 Dot Blot to Check Biotinylation of DNA 2.5 Biotin-Labeled DNA Fragment Pulldown and Sequencing Library Preparation 3 Methods 3.1 TSA Cell Labeling 3.1.1 TSA Labeling for Cells in Suspension Culture 3.1.2 TSA Labeling for Cells in Adherent Culture 3.2 Quality Control 1: Immunostaining and Microscopy 3.3 Genomic DNA Extraction and Sonication 3.4 Quality Control 2: Assaying DNA Biotinylation Levels with Dot Blots 3.5 Drosophila Spike-In DNA Preparation 3.6 Bead Pulldown of Biotin-Labeled DNA Fragments 3.7 DNA Library Preparation, Sequencing, and Data Processing 4 Notes References Chapter 9: The High-Salt Recovered Sequence-Sequencing (HRS-seq) Method: Exploring Genome Association with Nuclear Bodies 1 Introduction 2 Materials 3 Methods 3.1 Cell Nuclei Preparation (See Note 6) 3.2 Generation of Nuclear Halos by High-Salt Treatment 3.3 Digestion of Nuclear Halos 3.4 DNA Purification of StyI-Digested DNA from Soluble and Insoluble Fractions 3.5 Redigestion of Genomic DNA (See Note 10) 3.6 Sequencing Library Preparation 3.7 Bioinformatic Analyses of HRS-seq Data (See Note 13) 4 Notes References Part IV: Single-Cell Approaches Chapter 10: High-Throughput Preparation of Improved Single-Cell Hi-C Libraries Using an Automated Liquid Handling System 1 Introduction 2 Materials 3 Methods 3.1 Hi-C Preparation 3.2 Single-Cell Isolation and Library Preparation 3.3 Individual Library Purification (See Note 21) 3.4 Pooled Library Purification (See Note 21) 3.5 Library Quality Control and Sequencing 4 Notes References Chapter 11: Simultaneous Quantification of Spatial Genome Positioning and Transcriptomics in Single Cells with scDam&T-Seq 1 Introduction 2 Materials 2.1 Establishing Clonal Line 2.2 Bulk DamID2 2.3 scDam&T-seq 2.4 Sequencing Library Preparation 3 Methods 3.1 Creating a Clonal mESC Line Expressing Tir1 and the mAID-Dam-Lamin B1 Fusion Protein 3.2 Bulk DamID2 3.3 Preparing Single Cell Samples for scDam&T-seq 3.4 scDam&T-seq 3.5 Preparation of Illumina Sequencing Libraries 3.6 Raw Data Processing and Visualization 4 Notes References Part V: Visualizing Spatial Genome Organization Chapter 12: High-Throughput DNA FISH (hiFISH) 1 Introduction 2 Materials 2.1 Nick Translation for BAC and Fosmid Probes 2.2 Amplification and Preparation of OligoFISH Libraries (Adapted from) 2.3 Cell Culture in 384-Well Imaging Plate Format 2.4 DNA FISH in 384-Well Imaging Plate Format 2.5 Automated Image Acquisition and Analysis 3 Methods 3.1 Selection of Fluorescent Probe Types for hiFISH 3.2 Nick Translation for BAC and Fosmid Probes 3.3 Amplification and Preparation of OligoFISH Libraries (Adapted from) 3.3.1 Primary PCR Amplification and Aliquoting 3.3.2 Precipitation and Determination of Concentration of PCR Products 3.3.3 Secondary PCR Amplification 3.3.4 T7 In Vitro Transcription (IVT), Reverse Transcription (RT) and Alkaline Degradation of the RNA Strand 3.3.5 Final Cleanup 3.3.6 Dilution in Hybridization Buffer for FISH 3.4 Cell Culture in 384-Well Imaging Plate Format 3.4.1 Growing Adherent Cells in 384-Well Imaging Plates 3.4.2 Spinning Suspension Cells in 384-Well Imaging Plates 3.4.3 Growing Colony-Forming Cells on Plates 3.5 DNA FISH in 384-Well Plate Format (Fig. 2a) 3.6 Automated Image Acquisition Setup 3.7 Image Analysis 3.7.1 Image Analysis Pipeline Setup 3.7.2 Visual Inspection of Image Analysis Results as a Quality Control Step (See Note 33) 3.8 Statistical Analysis 3.8.1 Pairwise FISH Signal Distance Calculations 3.8.2 Determine Your Null Hypothesis: Experimentally Define What ``Colocalization´´ Means 3.8.3 Consider Effect Size vs. Statistical Significance, Consider Bootstrapping 4 Notes References Chapter 13: Versatile CRISPR-Based Method for Site-Specific Insertion of Repeat Arrays to Visualize Chromatin Loci in Living C... 1 Introduction 2 Materials 3 Methods 3.1 sgRNA Design and Cloning 3.2 Primer Design for Genomic PCR Amplification of the Homology Arms 3.3 Repair Cassette Cloning 3.4 Nickase-Mediated Knock-in 3.5 Primer Design for Genomic DNA PCR Screening 3.6 Clone Screening 3.7 Fixed-Cell Imaging for Verification of PCR-Positive Clones 3.8 Live-Cell Imaging of the Chromatin Loop Anchors 4 Notes References Part VI: Functionally Dissecting Chromatin Architecture Chapter 14: CLOuD9: CRISPR-Cas9-Mediated Technique for Reversible Manipulation of Chromatin Architecture 1 Introduction 2 Materials 2.1 sgRNA Vector Construction 2.2 Lentiviral Transduction 2.3 Western Blot 2.4 CLOuD9 Induced Dimerization and ChIP 2.5 CLOuD9 3C 3 Methods 3.1 sgRNA Vector Construction 3.2 Lentiviral Transduction 3.2.1 Suspension Cells 3.2.2 Adherent Cells 3.3 Western Blot 3.4 Induced Dimerization and Washout 3.5 CLOuD9 ChIP 3.6 CLOuD9 3C 4 Notes References Chapter 15: Acute Protein Depletion Strategies to Functionally Dissect the 3D Genome 1 Introduction 1.1 Targeted Protein Degradation to Study Early Molecular Events of 3D Genome Assembly 2 Systems for Targeted Protein Degradation 2.1 Acute Depletion of Endogenous Proteins 2.1.1 PROTACs 2.1.2 Anti/Nanobody Mediated Degradation 2.2 Acute Depletion Based on Endogenously Tagged Proteins 2.2.1 Shield 2.2.2 Small Molecule Assisted Shutoff (SMASh-Tag) 2.2.3 Auxin Inducible Degron (AID) System 2.2.4 Degradation Tag (dTAG) 3 Methods: General Considerations for Targeted Protein Degradation 3.1 Design of a Donor Template 3.2 Functionality of Fusion Proteins 3.3 Selection Strategies to Increase Knockin Efficiency 3.3.1 Antibiotic Resistance Markers 3.3.2 Fluorescent Proteins 3.4 Validation of Clones and Protein Functionality 3.4.1 PCR Screening of Edited Clones 3.4.2 Western Blotting for Functional Knockin 3.4.3 Sequencing for Correct Integration and Editing 4 Considerations for 3D Genome Applications 5 Future Directions References Index