The Nucleus

Preface
Contents
Contributors
Chapter 1: Live-Cell Imaging and Analysis of Nuclear Body Mobility
	1 Introduction
	2 Materials
		2.1 Cells and Cell Culture
		2.2 Microscopy
		2.3 Image Analysis Software
	3 Methods
		3.1 Cell Culture
		3.2 Induction of iPNB Formation (See Note 4)
		3.3 Live-Cell Imaging
		3.4 Image Analysis
	4 Notes
	References
Chapter 2: Laser Targeted Oligo Ligation (LTOL) to Identify DNA Sequences in the Vicinity of a Single Subnuclear Structure in ...
	1 Introduction
	2 Materials
		2.1 PEN (Polyethylene Naphthalate) Coverslips (See Subheading 3.1)
		2.2 Immunolabeling and Laser Targeting
		2.3 Oligo Ligation
		2.4 Microdissection and DNA Extraction and Amplification
	3 Methods
		3.1 Preparation of PEN Coverslips
		3.2 Immunolabeling Subnuclear Structures and Laser Targeting
		3.3 Oligo Ligation to DNA in the Targeted Region
		3.4 Laser Microdissection Pressure Catapulting of Single Cells and DNA Extraction and Amplification
	4 Notes
	References
Chapter 3: Visualizing Chromatin Modifications in Isolated Nuclei
	1 Introduction
	2 Materials
		2.1 Cells and Cell Culture
		2.2 Isolation of Nuclei
		2.3 Immunolabeling, Imaging, and Image Analysis
	3 Methods
		3.1 Isolation of Nuclei
		3.2 Labeling Nuclei
		3.3 Confocal Microscopy, Image Acquisition, and Analysis
	4 Notes
	References
Chapter 4: Dual-Color Metal-Induced Energy Transfer (MIET) Imaging for Three-Dimensional Reconstruction of Nuclear Envelope Ar...
	1 Introduction
	2 Materials and Methods
		2.1 MIET Substrate Preparation
		2.2 Cell Culture and Immunofluorescence
		2.3 MIET Measurements
		2.4 Fluorescence Lifetime Data Evaluation
		2.5 MIET Measurements and Data Evaluation
		2.6 MIET Calibration Curves and Axial Localization
		2.7 Quantum Yield Measurements
	3 Notes
	References
Chapter 5: Studying Proton Gradients Across the Nuclear Envelope
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Equipment and Software
	3 Methods
		3.1 Cells and Cell Growth
		3.2 Intracellular Loading of the pH Fluoroprobe SNARF-1
		3.3 Fluorescence Measurements of Cytosolic and Nuclear  pH
		3.4 In Situ Titration of the pH Fluoroprobe
		3.5 Data Analysis
		3.6 Other Experimental Approaches to Measure pH in Living Cells
	4 Notes
	References
Chapter 6: COMBinatorial Oligonucleotide FISH (COMBO-FISH) with Uniquely Binding Repetitive DNA Probes
	1 Introduction
	2 Materials
	3 Methods
		3.1 DNA Sequence Database Analysis and Probe Search
		3.2 Preparation of Oligonucleotide Probes
		3.3 Multicolor COMBO-FISH with Uniquely Binding Oligonucleotide Probes
		3.4 Combined COMBO-FISH and Immunostaining with Uniquely Binding Oligonucleotide Probes and Specific Antibodies
	4 Notes
	References
Chapter 7: Genome-Wide Mapping of UV-Induced DNA Damage with CPD-Seq
	1 Introduction
	2 Materials
		2.1 Growth and UV Irradiation of Yeast
		2.2 Human Cell Culture
		2.3 Isolation of Yeast Genomic DNA
		2.4 Isolation of Human Genomic DNA
		2.5 Reagents for CPD-seq Library Preparation
		2.6 Major Equipment for Library Preparation and Sequencing
	3 Methods
		3.1 Cell Growth, UV Irradiation, and Repair Incubation
			3.1.1 Growth and UV Irradiation of Yeast Cells
			3.1.2 Growth and UV Treatment of Human Fibroblasts
		3.2 Isolation of Genomic DNA
			3.2.1 Isolation of Yeast Genomic DNA
			3.2.2 Isolation of Genomic DNA from Human Cells
		3.3 DNA Sonication and Purification on AMPure XP Beads
		3.4 CPD-seq Library Preparation
		3.5 Bioinformatics Analysis of CPD-seq Data
	4 Notes
	References
Chapter 8: AP-Seq: A Method to Measure Apurinic Sites and Small Base Adducts Genome-Wide
	1 Introduction
	2 Materials
		2.1 Oxidative DNA Damage Treatment
		2.2 DNA Extraction and Processing
		2.3 8-oxoG-Specific Material
		2.4 Pulldown
		2.5 AP-Quant
		2.6 AP-qPCR
		2.7 AP-Seq: Library Preparation and Sequencing
		2.8 Software and Data for Core Data Processing
	3 Methods
		3.1 Experimental Design
		3.2 Preparation of Cells
		3.3 Genomic DNA Extraction and Processing
		3.4 Sample Processing for 8-oxoG
		3.5 Sample Processing for AP-Sites
		3.6 Pulldown of ARP-Tagged  gDNA
		3.7 AP-qPCR
		3.8 Library Preparation and Sequencing
		3.9 Core Data Processing
	4 Notes
	References
Chapter 9: Locus-Specific Chromatin Proteome Revealed by Mass Spectrometry-Based CasID
	1 Introduction
	2 Materials
		2.1 Choice of Biotin Ligase and Cloning Steps
		2.2 Generation of Stable BirA-dCas9-eGFP/gRNA Cell Lines
		2.3 Immunofluorescence Staining and Image Acquisition
		2.4 Biotin Pulldown and Sample Preparation for Mass Spectrometry
		2.5 LC-MS/MS Measurements
		2.6 General Equipment (Recommendations)
	3 Methods
		3.1 Choice of Biotin Ligase and Cloning Steps
		3.2 Generation of Stable BirA-dCas9-eGFP/gRNA Cell Lines
		3.3 Immunofluorescence Staining and Image Acquisition
		3.4 Biotin Pulldown and Sample Preparation for Mass Spectrometry
		3.5 LC-MS/MS Analysis
		3.6 Computational Analysis of MS  Data
	4 Notes
	References
Chapter 10: Methyl Adenine Identification (MadID): High-Resolution Detection of Protein-DNA Interactions
	1 Introduction
	2 Materials
		2.1 Generation of Cells Expressing M.EcoGII Constructs
		2.2 m6Adenine Immunofluorescence
		2.3 DNA Fluorescence In Situ Hybridization (FISH)
		2.4 Extraction of Genomic  DNA
		2.5 m6Adenine Immunoprecipitation (m6A-IP) Followed by High-Throughput Sequencing
		2.6 m6A Immunodot (m6A-Dotblot) Detection
		2.7 Telomere Purification by TeloCapture
	3 Methods
		3.1 Transfection of Phoenix Packaging Cells
		3.2 Transduction of Cells
		3.3 Culture of Transduced Cells and M.EcoGII Construct Induction Using Shield-1
		3.4 m6A Immunofluorescence
		3.5 DNA Fluorescence In Situ Hybridization (FISH) Following m6Adenine Immunofluorescence
		3.6 Isolation of Genomic DNA for m6A-IP-seq and m6A-Dotblots
		3.7 m6A-IP-seq Procedure
		3.8 m6A-Dotblots
		3.9 m6A-Dotblot on Isolated DNA Regions: Example of Telomere Purification by TeloCapture
	4 Notes
	References
Chapter 11: Optimized Detection of Protein-Protein and Protein-DNA Interactions, with Particular Application to Plant Telomeres
	1 Introduction
	2 Materials
		2.1 Bimolecular Fluorescence Complementation (BiFC)
			2.1.1 Solutions and Equipment
			2.1.2 Plants and Growth Conditions
			2.1.3 DNA Constructs
		2.2 Yeast Two-Hybrid System (Y2H)
			2.2.1 Solutions and Equipment
			2.2.2 DNA Constructs and Yeast Strains
		2.3 Co-immunoprecipitation (Co-IP) with Two or Three Proteins of Interest
			2.3.1 Solutions and Equipment
			2.3.2 In Vitro Transcription/Translation
		2.4 Chromatin Immunoprecipitation Assay (ChIP) for Low-Abundance Proteins
			2.4.1 Solutions and Equipment
	3 Methods
		3.1 Bimolecular Fluorescence Complementation (BiFC)
			3.1.1 DNA Constructs
			3.1.2 Isolation of A. thaliana Protoplasts
			3.1.3 Transfection of Protoplasts
		3.2 Yeast Two-Hybrid System (Y2H)
			3.2.1 DNA Constructs for Y2H
			3.2.2 Transformation of Yeast Cells
			3.2.3 Screening for Protein-Protein Interactions
		3.3 Co-immunoprecipitation (Co-IP) with Two or Three Proteins of Interest
			3.3.1 Transcription and Translation
			3.3.2 Co-immunoprecipitation
			3.3.3 Analysis of Radioactively Labeled Immunoprecipitates
		3.4 Chromatin Immunoprecipitation Assay (ChIP) for Low-Abundance Proteins
			3.4.1 Preparation of Plant Material and Chromatin Cross-Linking
			3.4.2 Isolation of Nuclei and Chromatin (see Note 35)
			3.4.3 Preparation of Immunosorbent Beads
			3.4.4 Immunoprecipitation
			3.4.5 Elution and Reversal of Cross-Links
	4 Notes
	References
Chapter 12: Macromolecular Crowding Measurements with Genetically Encoded Probes Based on Förster Resonance Energy Transfer in...
	1 Introduction
	2 Materials
		2.1 Synthetic Drop-Out Growth Medium Without Histidine
		2.2 Hyperosmotic Shock and Imaging
	3 Methods
		3.1 Yeast Strain and Sensor Choice
		3.2 Growing Yeast Cells and Expressing the Crowding Sensor
		3.3 Hyperosmotic Shock and Crowding Measurements
		3.4 Image Data Analysis
	4 Notes
	5 Requests for Materials
	References
Chapter 13: Analysis of a Nuclear Intrinsically Disordered Proteome
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Isolation of Nuclei
		2.3 Isolation of Proteins from Nuclei
		2.4 Enrichment of Intrinsically Disordered Proteins
		2.5 Sample Preparation for Mass Spectrometric Analysis
		2.6 LC-MS/MS Analysis
	3 Methods
		3.1 Cell Culture
		3.2 Preparation of Cell Nuclei
		3.3 Isolation of Proteins from Cell Nuclei
		3.4 Enrichment of Intrinsically Disordered Proteins
		3.5 Sample Preparation for Mass Spectrometric Analysis
		3.6 LC-MS/MS Analysis
		3.7 Analysis of Protein Disorder
		3.8 Gene Ontology Analysis
		3.9 Examples of Results
	4 Notes
	References
Chapter 14: Timing of Cytosine Methylation on Newly Synthesized RNA by Electron Microscopy
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Equipment
		2.3 Antibodies
	3 Methods
		3.1 Sample Preparation
		3.2 EM Immunocytochemistry
		3.3 Staining
		3.4 Analysis of Antibody Distribution
	4 Notes
	References
Chapter 15: The Nucleus of Intestinal Cells of the Bacterivore Nematode Caenorhabditis elegans as a Sensitive Sensor of Enviro...
	1 Introduction
	2 Materials
		2.1 Strains
		2.2 Reagents, Buffers, and Solutions
		2.3 Equipment
	3 Methods
		3.1 Culture and Synchronization of C. elegans
		3.2 Exposure of C. elegans to Environmental Pollutants
		3.3 Immobilization and Microscopy of FIB-1::GFP Reporter Worms
	4 Notes
	References
Index