CHROMOSOME ARCHITECTURE : methods and protocols.

Preface
Contents
Contributors
Chapter 1: A Next Generation of Advances in Chromosome Architecture
	1 Introduction
	References
Chapter 2: Single-Molecule Narrow-Field Microscopy of Protein-DNA Binding Dynamics in Glucose Signal Transduction of Live Yeas...
	1 Introduction
	2 Materials
		2.1 Fluorescently Labeled Yeast Strains
		2.2 Sample Preparation
		2.3 Narrow-Field Microscope
		2.4 Computational Analysis
	3 Methods
		3.1 Growing Yeast Strains
		3.2 Plasma Cleaning Coverslips
		3.3 Preparing Cell Samples
		3.4 Agarose Pad Preparation
		3.5 Obtaining Single-Molecule Data
		3.6 Tracking Single Molecules
		3.7 Analyzing Single-Molecule Trajectories
		3.8 Categorizing Tracks by Cell Compartment
	4 Notes
	References
Chapter 3: Convolutional Neural Networks for Classifying Chromatin Morphology in Live-Cell Imaging
	1 Introduction
		1.1 Chromatin Morphology Changes Throughout Cell´s Life Cycle
		1.2 Machine Learning Strategies for Cell-State Classification
		1.3 Exploratory Visualization of CNN Feature Embedding
	2 Materials
	3 Methods
		3.1 Annotating Training Images Using Napari
		3.2 Training a CNN for Chromatin Morphology Classification
			3.2.1 Training the CNN on a Train  Set
			3.2.2 Evaluating the CNN on a Validation  Set
		3.3 Using the CNN for Chromatin Morphology Classification on Unseen  Data
			3.3.1 Using the Trained CNN for Inference on a Test  Set
			3.3.2 Visualizing the CNN Feature Embedding
	4 Notes
	References
Chapter 4: Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SM...
	1 Introduction
	2 Materials
		2.1 Growth Media
		2.2 Slides and Microscope
	3 Methods
		3.1 Preparation of Bacterial Cultures for Microscopy
		3.2 Preparation of Microscopy Slide
		3.3 Microscopy
		3.4 Image Analysis
	4 Notes
	5 Conclusions
	References
Chapter 5: Atomic Force Microscopy of DNA and DNA-Protein Interactions
	1 Introduction
	2 Materials
		2.1 General Materials
		2.2 AFM Cantilevers
		2.3 Buffer Solutions
		2.4 DNA Substrates
	3 Methods
		3.1 Preparation of Mica Substrate
		3.2 Methods for DNA Adsorption on a Mica Substrate for AFM Imaging in Fluid
			3.2.1 DNA Adsorption Using Divalent Cations
			3.2.2 DNA Adsorption Using  PLL
			3.2.3 DNA Adsorption Using PLL-b-PEG
		3.3 Pre-Imaging Setup for High-Resolution AFM in Fluid
		3.4 Optimizing AFM Imaging in PFT for High-Resolution AFM Imaging on  DNA
		3.5 Methods for DNA-Protein Imaging by AFM in Fluid
	4 Notes
	References
Chapter 6: Live-Cell Visualization of DNA Transfer and Pilus Dynamics During Bacterial Conjugation
	1 Introduction
	2 Materials
		2.1 Medium for Cell Culture and Dye Reagents
		2.2 Agarose´s Pad and Microfluidics Plates
		2.3 Image Analysis Suite
	3 Methods
		3.1 Cysteine Substitution Mutants for Maleimide Bioconjugation
		3.2 Labeling of the Conjugative Pilus
		3.3 Visualization of Plasmid DNA Transfer
		3.4 Live-Cell imaging on Agarose-Mounted Slides
		3.5 Live-Cell Imaging Within Microfluidic Chambers
		3.6 Imaging Acquisition
		3.7 Image Analysis
	4 Notes
	References
Chapter 7: Transverse Magnetic Tweezers Allowing Coincident Epi-Fluorescence Microscopy on Horizontally Extended DNA
	1 Introduction
	2 Materials
		2.1 Buffers and Special Reagents
		2.2 Preparation of Microspheres Labeled with Anti-Digoxigenin (PAG-AD-MS)
		2.3 Preparation of Oxygen Scavenger System
		2.4 Preparation of Microscopy Substrates
		2.5 Preparation of Horizontal DNA Tethers
		2.6 Combined Magnetic Tweezers and Epi-Fluorescence Microscope
	3 Methods
		3.1 Preparation of Microspheres Labeled with Anti-Digoxigenin
		3.2 Preparation of Oxygen Scavenger System
		3.3 Preparation of Microscopy Substrates
		3.4 Preparation of Horizontal DNA Tethers
		3.5 Manipulation of DNA-Tethered Superparamagnetic Microspheres
	4 Notes
	References
Chapter 8: Atomistic Molecular Dynamics Simulations of DNA in Complex 3D Arrangements for Comparison with Lower Resolution Str...
	1 Introduction
		1.1 Overview of Simulation Protocols
		1.2 Computer Hardware, Software, and MD Force Fields
		1.3 Analysis Tools of DNA Simulations for Comparison with Experiments
	2 Materials
		2.1 MD Simulation, Analysis, and Visualization Software
		2.2 AMBERTOOLS Modules
		2.3 Source Files
	3 Methods
		3.1 Molecular Dynamics Simulations
		3.2 Conformational Analysis
	4 Notes
	References
Chapter 9: Replication Labeling Methods for Super-Resolution Imaging of Chromosome Territories and Chromatin Domains
	1 Introduction
	2 Materials
		2.1 Cell Culture and Labeling
		2.2 Sample Staining, Fixation, and Microscopy
	3 Methods
		3.1 F-ara-EdU Labeling of Individual Chromosome Territories
		3.2 Replication Domains and Origins for Fixed/Live Samples
		3.3 Differential Labeling of Sister Chromatids for Fixed Samples (Fig. 5)
	4 Notes
	References
Chapter 10: DNA-Protein Interactions Studied Directly Using iSCAT Imaging of GNP-Tagged Proteins
	1 Introduction
	2 Materials
		2.1 Interferometric Scattering Microscope
		2.2 Digoxigenin-Labeled Oligonucleotides
		2.3 5x ABC Buffer
		2.4 ABT Buffer
		2.5 mPEG Solution
		2.6 1x TE Buffer
		2.7 Anti-digoxigenin Solution
		2.8 Flow Cells
	3 Methods
		3.1 Slide and Coverslip Preparation
		3.2 Plasma Cleaning
		3.3 Slide and Coverslip Silanization
		3.4 Flow Cell Construction
		3.5 Calibration Curve Data Collection
		3.6 Annealing Oligonucleotides
		3.7 Experiment Setup
		3.8 Image Acquisition
		3.9 Image Processing
		3.10 Calibration Curve
		3.11 Data Analysis
	4 Notes
	References
Chapter 11: Escherichia coli Chromosome Copy Number Measurement Using Flow Cytometry Analysis
	1 Introduction
	2 Materials
	3 Methods
		3.1 Standard Strains
		3.2 dnaAts Mutants
		3.3 Staining Cells for Flow Cytometry
	4 Notes
	References
Chapter 12: Dynamics of Bacterial Chromosomes by Locus Tracking in Fluorescence Microscopy
	1 Introduction to Live-Cell Locus Tracking at High Frame Rate
	2 Materials
		2.1 Bacteria Strains and Reagents
		2.2 Microscopy
	3 Methods
		3.1 Sample Preparation
		3.2 Video Acquisition
		3.3 Image Analysis
		3.4 Considerations on Precision and System-Specific Aspects of the Localization Data
		3.5 Choice of the Protein Fusion to Label Foci
		3.6 Considerations on the Correlation of Loci Motility to Intensity
		3.7 Cellular Density Estimation
		3.8 Control of Cell Lysis
		3.9 Refraction Index Estimation
	4 Notes
	References
Chapter 13: Measuring Nuclear Mechanics with Atomic Force Microscopy
	1 Introduction
	2 Materials
		2.1 Atomic Force Microscope
		2.2 Cantilevers
		2.3 Coverslip cleaning
	3 Methods
		3.1 Cell Seeding: Full-Adhesion Measurements
		3.2 Cell Seeding: Initial Adhesion Measurements
		3.3 Nuclei Seeding: Isolated Nuclei
		3.4 Cantilever Calibration
		3.5 Mechanical Measurements of the Nucleus
		3.6 Data Analysis: Calculation of the Young´s Modulus E
	4 Notes
	References
Chapter 14: Copy Number Analysis of the Yeast Histone Deacetylase Complex Component Cti6 Directly in Living Cells
	1 Introduction
	2 Materials
		2.1 Yeast Strains
		2.2 Super-Resolution Microscopy
	3 Methods
		3.1 Fluorescent Labeling of the Cti6 Protein
		3.2 Conformation PCR
		3.3 Preparing Cells for Microscopy
		3.4 Preparing Microscopy Slides Using 125 μL Gene Frame
		3.5 Data Acquisition
		3.6 Copy Number Analysis
	4 Notes
	References
Chapter 15: Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells
	1 Introduction
	2 Materials
		2.1 Lambda Red Recombination
		2.2 Cell Culture, Labeling, and Slide Preparation
		2.3 Microscope
		2.4 Data Analysis
	3 Methods
		3.1 Lambda Red Integration to Generate an Endogenous Fluorescent Fusion
		3.2 Cell Culture
		3.3 Microscopy
		3.4 Reconstructing a Super-Resolution Map of Localizations
		3.5 Short Exposure Times to Quantify Diffusion
		3.6 Long Exposures to Quantify Binding Kinetics
		3.7 Clustering Analysis
	4 Notes
	References
Chapter 16: Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localization Microsco...
	1 Introduction to the Role of Live-Cell Single-Molecule Localization Microscopy
	2 Materials
		2.1 SMLM Microscope Configuration
			2.1.1 Sample Illumination
			2.1.2 Sample Detection
		2.2 Labeling Mammalian Cells
			2.2.1 Fluorophore Choice
			2.2.2 Designing Expression Vectors
	3 Methods
		3.1 Sample Preparation
		3.2 Microscope Setup
		3.3 Experimental Design for Single-Molecule Imaging and Tracking
			3.3.1 Short-Exposure Tracking of Single-Protein Diffusion and Binding to Chromatin
			3.3.2 Long-Exposure Tracking of Single Chromatin-Bound Proteins for Probing Chromatin Diffusion
			3.3.3 Time-Lapse Experiments for Determining Residence Time
			3.3.4 Structural Imaging
		3.4 Data Analysis
			3.4.1 Quantifying Numbers of Molecules
			3.4.2 Extracting Clustering Parameters
			3.4.3 Extracting Biophysical Parameters from SPT
			3.4.4 Residence Time
			3.4.5 Using Photobleaching Rates to Analyze Single-Molecule FRET Trajectories
			3.4.6 Analyzing Images with High Density of Localizations
	4 Notes
	References
Chapter 17: The End Restraint Method for Mechanically Perturbing Nucleic Acids In Silico
	1 Introduction
		1.1 DNA Mechanics In Vivo
		1.2 Experimental Methods
		1.3 Simulation Approaches
		1.4 Advantages of Our Approach
	2 Materials
		2.1 Molecular Dynamics Software
		2.2 Visualization Software
		2.3 Analysis Utilities
		2.4 Bespoke Scripts and Input Files
	3 Methods
		3.1 Initial System Preparation
		3.2 Applying Torsion
		3.3 Equilibration
		3.4 Running the Umbrella-Sampling Simulations
		3.5 Analysis Strategies
	4 Notes
	References
Chapter 18: Visualizing the Replisome, Chromosome Breaks, and Replication Restart in Bacillus subtilis
	1 Introduction
	2 Materials
		2.1 Growth of B. subtilis.
		2.2 General Microscopy
	3 Methods
		3.1 Growth Conditions for Visualizing the Nucleoid, Origins, and Replisome in B. subtilis
		3.2 Growth Conditions for DNA Damage Assays
		3.3 Growth Conditions for Replication Restart Assays
		3.4 Preparation of Multi-Spot Microscope Slides
		3.5 Preparation of Microscope Slide (Gene Frame)
	4 Notes
	References
Chapter 19: High-Throughput Imaging of Bacillus subtilis
	1 Introduction
	2 Materials
		2.1 Media and Equipment for the Growth of B. Subtilis
		2.2 Components for Imaging
	3 Methods
		3.1 Exponential Growth of Cells
		3.2 Prepare 96-Well Pedestals
		3.3 Mounting B. subtilis Strains on the Agarose Pedestals
		3.4 High-Throughput Imaging of B. subtilis Strains Using Wide-Field Fluorescence Microscopy
		3.5 Option 1: Setting Software for Semiautomated Image Acquisition
		3.6 Option 2: Setting Software for Automated Image Acquisition
		3.7 Characterization of Mutant Phenotypes by Quantitative Image Analysis
	4 Notes
	References
Chapter 20: Measuring Nuclear Organization of Proteins with STORM Imaging and Cluster Analysis
	1 Introduction
	2 Materials
		2.1 Etch Solution
		2.2 Imaging Buffer
		2.3 Coverslip Preparation
	3 Methods
		3.1 Cell Preparation
		3.2 Immunofluorescence
		3.3 Imaging Sample Preparation
		3.4 STORM Image Acquisition
		3.5 STORM Image Processing
		3.6 Cluster Analysis of STORM  Data
	4 Notes
	References
Chapter 21: Single-Molecular Quantification of Flowering Control Proteins Within Nuclear Condensates in Live Whole Arabidopsis...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Live Arabidopsis Roots for Microscopy
		3.2 Preparation of a Standard Yeast Sample for MICROSCOPY
		3.3 Preparation of an AiryScan Alignment Sample
		3.4 Optional: Slimfield Imaging of the Standard Sample
		3.5 AiryScan Imaging of the Standard and Root Samples
		3.6 Single-Molecule Brightness Using Literature Estimates
		3.7 Optional: Verification of Single-Molecule Calibration Using Slimfield Imaging
		3.8 Applying the Calibration to the Analysis of AiryScan Plant Root Images
	4 Notes
		4.1 Preparation of Live Roots for Investigation
		4.2 Preparation of the Standard Yeast Sample
		4.3 Preparation of an AiryScan Alignment Sample
		4.4 Slimfield Imaging
		4.5 AiryScan imaging
		4.6 Single-Molecule Calibration, Verification, and Implementation
	References
Index