Bacterial Regulatory RNA: Methods and Protocols (Methods in Molecular Biology, 2741)

Preface/Summary
Contents
Contributors
Part I: sRNA Discovery
	Chapter 1: RNA Extraction from Gram-Positive Bacteria Membrane Vesicles Using a Polymer-Based Precipitation Method
		1 Introduction
		2 Materials
		3 Methods
			3.1 Concentration of MVs from Culture Supernatants
			3.2 Precipitation of MVs
			3.3 Isolation of RNA from MVs with the miRNeasy Kit
		4 Notes
		References
	Chapter 2: Extraction and Purification of Outer Membrane Vesicles and Their Associated RNAs
		1 Introduction
		2 Materials
			2.1 Bacterial Growth
			2.2 OMVs Extraction
				2.2.1 Ultracentrifugation
				2.2.2 Ultrafiltration
				2.2.3 Ion-Exchange-Based Columns
			2.3 OMVs Purification
			2.4 OMVs Analysis
			2.5 RNA Extraction and Analysis
				2.5.1 RNA Extraction Methods
				2.5.2 Quality Control
				2.5.3 RNA Analysis
		3 Methods
			3.1 OMVs Extraction
				3.1.1 Bacterial Growth
				3.1.2 Ultracentrifugation
				3.1.3 Ultrafiltration
				3.1.4 Centrifugation Steps
			3.2 OMVs Purification
				3.2.1 Density Gradient Purification
			3.3 OMVs Analysis
				3.3.1 OMVs Concentration
					FM1-43 Dosage
					ZetaSizer
					Nanoparticles Tracker Analyzer (NTA)
					Bradford Assay
				3.3.2 OMVs Observations
					Transmission Electron Microscopy (TEM)
					SDS-PAGE Electrophoresis
			3.4 Extraction of  RNAs
				3.4.1 RNA Extraction Methods
				3.4.2 Quality Control
				3.4.3 RNA Analysis
		4 Notes
		References
	Chapter 3: Analysis of Phage Regulatory RNAs: Sequencing Library Construction from the Fraction of Small Prokaryotic RNAs Less...
		1 Introduction
		2 Materials
			2.1 Reagents to Be Supplied by User
		3 Methods
			3.1 Isolation of the Bacterial sRNAs Using PureLink miRNA Isolation Kit (Invitrogen)
			3.2 DNA Digestion Using TURBO DNA-free Kit (Invitrogen)
			3.3 RNA Precipitation and Concentration
			3.4 Polyacrylamide Gel Electrophoresis (PAGE) of the Isolated Small RNA Molecules
			3.5 Elution of sRNAs Less Than 50 nt in Length from the Gel Slice
			3.6 Library Preparation Using SMARTer smRNA-Seq Kit for Illumina (Clontech Laboratories)
			3.7 Library Validation and Sequencing
		4 Notes
		References
	Chapter 4: Discovering Novel Bacterial Small RNA by RNA-seq Analysis Toolkit ANNOgesic
		1 Introduction
		2 Installation Guidelines
			2.1 Docker
			2.2 Singularity
			2.3 pip3
		3 Methods
			3.1 Arguments
			3.2 Download the Input Files
			3.3 Create an ANNOgesic Project Folder
			3.4 Place the Input Files in ANNOgesic Analysis Folders
			3.5 Detect Transcripts
			3.6 Identification of Factor-Independent Terminators (Optional)
			3.7 Download sRNA and nr Databases (Optional)
			3.8 Detection of sRNA
			3.9 Outputs of sRNA Prediction
				3.9.1 sRNA Annotations and Their Scores
				3.9.2 Secondary Structures and Sequences of the sRNA Candidates
				3.9.3 BLAST Results (for Reference)
				3.9.4 Plots
		4 Result Interpretation and Experimental Validation
		5 Trouble Shooting Guide
		6 Note
		References
Part II: sRNA Functions
	Chapter 5: Ribosome Profiling Methods Adapted to the Study of RNA-Dependent Translation Regulation in Staphylococcus aureus
		1 Introduction
		2 Materials
			2.1 S. aureus Cultures
			2.2 Cell Harvesting and Lysis
			2.3 Preparation of Total RNA
			2.4 Monosome/Polysome Enrichment and Buffer Exchange
			2.5 MNase Treatment
			2.6 Polysome Analysis and Monosome Isolation
			2.7 Hot Phenol RNA Extraction from Monosomes
			2.8 Purification of Ribosome Protected Fragments
		3 Methods
			3.1 S. aureus Cultures (to Be Performed in an L2 Laboratory)
			3.2 Cell Harvesting and Lysis (to Be Performed in an L2 Laboratory)
				3.2.1 Ice Bath Protocol
				3.2.2 Flash-Freezing Protocol
			3.3 Preparation of Total RNA for Transcriptomics Analysis
			3.4 Monosome/Polysome Enrichment and Buffer Exchange
			3.5 MNase Treatment
			3.6 Polysome Analysis and Monosome Isolation
			3.7 Hot Phenol RNA Extraction from Monosomes
			3.8 Purification of Ribosome-Protected Fragments
			3.9 Library Preparation
			3.10 Data Analysis
			3.11 Visualization and Data Interpretation with Ribo-RET Prediction Explorer
		4 Notes
		References
	Chapter 6: CRISPR Interference-Based Functional Small RNA Genomics
		1 Introduction
		2 Materials
			2.1 Design of the Guide Library
			2.2 Cloning of the Guide Library
			2.3 CRISPRi Screen
		3 Methods
			3.1 OPTIONAL: Compilation of the List of Targetable Sequences
			3.2 OPTIONAL: Identification of the Most Abundant PAM
			3.3 Design of the gRNA Library
			3.4 Cloning of the gRNAs
			3.5 OPTIONAL: Assembly and Cloning of CRISPR Arrays
			3.6 Transformation into the Donor Strain E. coli S17-1
			3.7 Conjugation into B. thetaiotaomicron
			3.8 Growth of the Library Under Selecting Conditions (CRISPRi Screen)
			3.9 gDNA Extraction
			3.10 PCR Amplification
			3.11 Library Preparation and Sequencing
			3.12 Data Analysis
		4 Notes
		References
	Chapter 7: Investigation of sRNA-mRNA Interactions in Bacillus subtilis In Vivo
		1 Introduction
			1.1 Application of Two-Plasmid Systems, Reporter Gene Fusions, and Chromosomal Deletions, Insertions, or Mutations to Study th...
			1.2 Determination of Expression Profiles, Half-Lives, and Intracellular Concentrations of sRNA and mRNA
			1.3 In Vivo Analysis of RNA Chaperones That Promote the sRNA-mRNA Interaction
		2 Materials
			2.1 Two-Plasmid Systems, Reporter Gene Fusions, and Chromosomal Deletions, Insertions, or Mutations to Study the Effect of sRN...
				2.1.1 Growth Media
				2.1.2 Vector, PCR Fragment Preparation, and Cloning
				2.1.3 Bacterial Strains
				2.1.4 Transformation and Selection
				2.1.5 β-galactosidase Measurements
				2.1.6 LFH Materials
			2.2 Northern Blotting
				2.2.1 RNA Isolation
				2.2.2 PAAGE and Tank Blotting
				2.2.3 Agarose Gels and Capillary Blotting
				2.2.4 Preparation of Riboprobes/Oligo-DNA Probes (See Chapter 8, Subheading 2.1.1)
				2.2.5 Prehybridization, Hybridization of the Probe, and Detection
			2.3 Two-Step Quantitative Real-Time PCR
				2.3.1 DNase I Treatment
				2.3.2 Quantitative cDNA Synthesis
				2.3.3 qRT-PCR
			2.4 In Vivo Analysis of RNA Chaperones That Promote the sRNA-mRNA Interaction
		3 Methods
			3.1 Two-Plasmid Systems, Reporter Gene Fusions, and Chromosomal Deletions, Insertions, or Mutations to Study the Effect of sRN...
				3.1.1 Cloning of Wild-Type or Mutated sRNA Gene into a Plasmid Vector (See Note 1)
				3.1.2 E. coli Transformation and Selection of Correct Clones
				3.1.3 Bacillus subtilis Transformation
				3.1.4 Long-Flanking Homology (LFH)-PCR
				3.1.5 Construction of Compensatory Mutations to Confirm Base-Pairing Interactions (See Notes 15, 16, 17, and 18)
				3.1.6 Construction of Transcriptional or Translational lacZ Reporter Gene Fusions to Demonstrate Effects of an sRNA on Transla...
			3.2 Determination of Expression Profiles, Half-Lives, and Intracellular Concentrations of sRNA and mRNA
				3.2.1 Isolation of Total RNA
				3.2.2 Separation of sRNA and Short Target mRNA in Polyacrylamide Gels and Subsequent Tank Blotting
				3.2.3 Separation of Target mRNAs >1 kb in Agarose Gels and Subsequent Capillary Blotting
				3.2.4 Preparation of Riboprobes and Oligo-DNA Probes
				3.2.5 Prehybridization, Hybridization, and Exposure
			3.3 Two-Step Quantitative Real-Time PCR
				3.3.1 DNase I Treatment
				3.3.2 Quantitative cDNA Synthesis
				3.3.3 qRT-PCR
			3.4 In Vivo Analysis of RNA Chaperones That Promote the sRNA-mRNA Interaction
		4 Notes
		References
	Chapter 8: In Vitro Methods for the Investigation of sRNA-mRNA Interactions in Bacillus subtilis
		1 Introduction
			1.1 In Vitro Synthesis and Purification of Labeled or Unlabeled RNA from Denaturing Polyacrylamide Gels
			1.2 Investigation of RNA-RNA Interactions by Electrophoretic Mobility Shift Assay (EMSA)
			1.3 Determination of Secondary Structures of the sRNA and the sRNA/Target RNA Complex
			1.4 Analysis of RNA Chaperones That Promote the sRNA-mRNA Interaction
		2 Materials
			2.1 In Vitro Synthesis and Purification of Labeled or Unlabeled RNA from Denaturing Polyacrylamide Gels
				2.1.1 In Vitro Synthesis of RNA
				2.1.2 Polyacrylamide Gel (PAAG) Preparation
			2.2 Investigation of RNA-RNA Interactions by Electrophoretic Mobility Shift Assay (EMSA)
				2.2.1 In Vitro Synthesis of Internally Labeled RNA
				2.2.2 In Vitro Synthesis and Purification of 5′ Labeled RNA
				2.2.3 Electrophoretic Mobility Shift Assay (EMSA)
			2.3 Determination of Secondary Structures of the sRNA and the sRNA/Target RNA Complex
				2.3.1 Enzymatic RNA Structure Probing
				2.3.2 Chemical RNA Secondary Structure Probing
				2.3.3 Chemical Probing with DMS (Dimethyl Sulfate)
				2.3.4 Chemical Probing with CMCT (1-Cyclohexyl-3-2-Morpholinoethyl Metho-p-Toluene Carbodiimide Sulfonate)
				2.3.5 Chemical Probing with Pb2+
				2.3.6 PAAG for Chemical Probing of RNA Secondary Structure
			2.4 Analysis of RNA Chaperones That Promote the sRNA-mRNA Interaction
				2.4.1 DRaCALA (Differential Radial Capillary Action of Ligand Assay)
				2.4.2 EMSAs for Binding of an RNA Chaperone to an RNA
				2.4.3 EMSAs to Study the Effect of an RNA Chaperone on an RNA-RNA Interaction
				2.4.4 RNA Secondary Structure Probing in RNA-protein Complexes
		3 Methods
			3.1 In Vitro Synthesis and Purification of Labeled or Unlabeled RNA from Denaturing Polyacrylamide Gels
				3.1.1 In Vitro Synthesis of the RNA
				3.1.2 RNA Purification by PAAG Electrophoresis and Subsequent Elution
				3.1.3 In Vitro Synthesis of Internally Labeled RNA
				3.1.4 Separation on a 6% Denaturing PAAG and Elution of the Labeled RNA
				3.1.5 Sephadex G-50 Column Preparation and Probe Purification (See Note 8, Fig. 1b)
				3.1.6 In Vitro Synthesis and Purification of 5′ Labeled RNA
				3.1.7 Dephosphorylation of the RNA
				3.1.8 Labeling with 32P-[γ-ATP]
			3.2 Investigation of RNA-RNA Interactions by Electrophoretic Mobility Shift Assay (EMSA)
				3.2.1 In Vitro Synthesis of sRNA and Target RNA and Labeling of One Interaction Partner
				3.2.2 Binding Reaction
				3.2.3 Gel Separation of Free RNA and Duplex
				3.2.4 Determination of the Kd of the RNA-RNA Complex
				3.2.5 Localization of the Minimal Inhibitory Sequence of an sRNA
				3.2.6 Determination of the Apparent Binding Rate Constant kapp
			3.3 Determination of Secondary Structures of the sRNA and the sRNA/Target RNA Complex
				3.3.1 Enzymatic Secondary Structure Probing of RNA
				3.3.2 RNase and Nuclease S1 Cleavage Reactions
				3.3.3 Preparation of T1 Ladder
				3.3.4 Preparation of Alkaline Ladder
				3.3.5 Determination of the RNA Secondary Structure After Enzymatic Cleavage (See Notes 24-26)
				3.3.6 Secondary Structure Probing of RNA-RNA Complexes
				3.3.7 Chemical Probing of RNA by Modification with DMS, CMCT, or Pb2+
				3.3.8 Chemical Probing of RNA by Modification with DMS
				3.3.9 Chemical Probing of RNA by Modification with CMCT
				3.3.10 Chemical Probing of RNA by Modification with Pb2+
				3.3.11 Determination of the Secondary Structure After Chemical Modification
			3.4 Analysis of RNA Chaperones That Promote the sRNA-mRNA Interaction
				3.4.1 DRaCALA (Differential Radial Capillary Action of Ligand Assay)
				3.4.2 RNA-Protein EMSA
				3.4.3 Applications of RNA-Protein EMSA
				3.4.4 RNA-RNA-Protein EMSA
				3.4.5 Evaluation of RNA-RNA-Protein EMSA
				3.4.6 RNA Secondary Structure Probing in RNA-protein Complexes
				3.4.7 Evaluation of Changes in the Secondary RNA Structure by RNA Chaperone Binding
		4 Notes
		References
	Chapter 9: RNA Double-Helix Hybridization Measured by Fluorescence Correlation Spectroscopy
		1 Introduction
			1.1 RNA Double-Strand Hybridization
			1.2 FCS Theory
		2 Materials
			2.1 ssRNAs
			2.2 Hybridization
			2.3 Fluorescence Correlation Spectroscopy (FCS)
			2.4 Software
		3 Methods
			3.1 Hybridization Assay
			3.2 Performance of FCS Measurement
			3.3 Data Analysis
		4 Notes
		References
	Chapter 10: New Perspectives on Crosstalks Between Bacterial Regulatory RNAs from Outer Membrane Vesicles and Eukaryotic Cells
		1 Introduction
		2 Regulatory sRNAs Carried by Membrane Vesicles
			2.1 sRNAs Carried by OMVs
				2.1.1 Crosstalk Between Host Immune System and OMV-Associated sRNAs
			2.2 sRNAs Carried by EVs or Other Membrane Vesicles
		3 Bacterial sRNAs Regulating Gene Expression in Plant Cells
		4 Future Perspectives
		References
	Chapter 11: Experimental Validation of RNA-RNA Interactions by Electrophoretic Mobility Shift Assay
		1 Introduction
		2 Materials
			2.1 DNA Template
			2.2 In Vitro Transcription and Purification of RNA
			2.3 Labeling of RNA
			2.4 EMSA
		3 Methods
			3.1 Design of Mutant Derivatives
			3.2 Design and Preparation of DNA Template
			3.3 In Vitro Transcription and Purification of RNAs
			3.4 Labeling of  RNAs
			3.5 EMSA
		4 Notes
		References
	Chapter 12: Dynamics and Function of sRNA/mRNAs Under the Scrutiny of Computational Simulation Methods
		1 Simulations
			1.1 Theoretical Framework
			1.2 The MM Force Fields
			1.3 Free Energy Landscapes and Conformational Ensembles
			1.4 Challenges for RNA MD Simulations
		2 Dynamical Characteristics of RNAs
			2.1 Ensemble Modularity in RNA
			2.2 Hierarchy of RNA Free Energy Landscapes
		3 MD Simulations Protocols
			3.1 Packages to Perform MD Simulations
			3.2 Setting Up the Model
			3.3 Minimisation, Heating, and Equilibration
			3.4 Production
			3.5 Enhanced Sampling
		4 Advances in RNA Simulations
			4.1 Benchmark Applications
			4.2 Applications to Regulatory sRNAs
		5 Conclusions
		6 Movie Legend
		References
	Chapter 13: Analysis of sRNAs and Their mRNA Targets in Sinorhizobium meliloti: Focus on Half-Life Determination
		1 Introduction
			1.1 Translation Inhibition as an Independent Signal for the Tryptophan Attenuator
			1.2 Considerations of Methods for RNA Isolation and RNA Stability Determination
		2 Materials
			2.1 Cultivation and Harvest
			2.2 RNA Isolation with Spike-In Transcript, Using RNeasy Mini Columns
			2.3 DNase Treatment
			2.4 RT-qPCR
		3 Methods
			3.1 Cultivation and Harvest
			3.2 RNA Isolation (According to the RNeasy Mini Kit Protocol) and Spike-In
			3.3 DNase Treatment
			3.4 RT-qPCR
			3.5 Cq Determination
			3.6 Primer Efficiency Evaluation
			3.7 Evaluation of mRNA Half-Life
		4 Notes
		References
	Chapter 14: Evaluation of 5′-End Phosphorylation for Small RNA Stability and Target Regulation In Vivo
		1 Introduction
		2 Materials
			2.1 Strain and Plasmid Constructions
			2.2 Cultures for Determination of (s)RNA Steady-State Levels
			2.3 Cultures for (s)RNA Half-Life Determinations
			2.4 Total RNA Extraction
			2.5 Denaturing Urea-Polyacrylamide Gel Electrophoresis for Separation of Total  RNA
			2.6 Northern Blotting
			2.7 Stripping the Nylon Membrane for Probe Removal
			2.8 Generation of DIG-Labeled RNA Probes
		3 Methods
			3.1 Construction of the Test Strain and Recombinant Plasmids for Release of sRNAx
			3.2 Cultivation of Bacteria for Determination of (s)RNA Steady-State Levels
			3.3 Cultivation of Bacteria for Determination of (s)RNA Decay Rates
			3.4 Extraction of Total RNA from the Bacterial Pellets
			3.5 Denaturing Urea Polyacrylamide Gel Electrophoresis
			3.6 Northern Blotting
			3.7 Stripping the Nylon Membrane for Detection of Distinct RNA Species
			3.8 Generation of DIG-Labeled RNA Probes by In Vitro Transcription
		4 Notes
		References
	Chapter 15: In-Gel Cyanoethylation for Pseudouridines Mass Spectrometry Detection of Bacterial Regulatory RNA
		1 Introduction
		2 Materials
			2.1 Instruments and Equipment
			2.2 Gel Electrophoresis
			2.3 In-Gel Cyanoethylation of ψ and RNase Digestion
			2.4 Nano Liquid Chromatography
		3 Methods
			3.1 RNA Purification by Gel Electrophoresis
			3.2 In-Gel Cyanoethylation and RNase Digestion
			3.3 RNA Digest Products Desalting
			3.4 nanoLC-MS/MS
				3.4.1 Nano Liquid Chromatography
				3.4.2 Mass Spectrometry Analysis
			3.5 Data Analysis
		4 Notes
		References
Part III: sRNA Interactome
	Chapter 16: Directed Screening for sRNA Targets in E. coli Using a Plasmid Library
		1 Introduction
		2 Materials
			2.1 Molecular Cloning of sRNA Genes
			2.2 mRNA Translational Fusion Construction
			2.3 Library Screening
		3 Methods
			3.1 Molecular Cloning of sRNA Genes
			3.2 mRNA Translational Fusion Construction
				3.2.1 Insertion DNA Preparation
				3.2.2 Recombineering (Fig. 3b)
			3.3 Library Screening
				3.3.1 TSS Transformation (Fig. 4)
				3.3.2 β-Gal Assay Using 96-Well Microplates (Fig. 4)
		4 Notes
		References
	Chapter 17: Defining Bacterial RNA-RNA Interactomes Using CLASH
		1 Introduction
		2 Materials
			2.1 Strains
			2.2 Growth Medium
			2.3 Buffers
			2.4 Solutions
			2.5 Enzymes and Reagents (See Notes 1 and 2)
			2.6 Consumables
			2.7 Adapters and Primers
			2.8 Equipment
		3 Method
			3.1 Experimental Procedure
				3.1.1 Cell Growth
				3.1.2 Cell Lysis
				3.1.3 RNP-HTF Immunoprecipitation with Anti-FLAG Magnetic Beads
				3.1.4 TEV Digestion of HTF Epitope
				3.1.5 RNase Trimming of Transcripts Cross-Linked to the RBP
				3.1.6 Ni-NTA Affinity Purification of RNP-His6 and 3′ Dephosphorylation of Covalently Bound RNAs
				3.1.7 Phosphorylation of the 5′ Ends of Cross-Linked RNAs with Radioactive 32P
				3.1.8 On-Bead Ligation of the 5′ Linker to the Cross-Linked RNAs
				3.1.9 On-Bead Ligation of the App-PE Linker to the 3′ End of the RNAs
				3.1.10 Elution of RBP and Cross-Linked RNAs from Ni-NTA Agarose Resin
				3.1.11 Trichloroacetic Acid (TCA) Precipitation of RNPs
				3.1.12 Proteinase K Digestion of the Purified RBP
				3.1.13 Extraction of Cross-Linked RNAs
				3.1.14 Reverse Transcription of Purified RNAs
				3.1.15 Purification of cDNA Library
				3.1.16 Amplification of cDNA Library
				3.1.17 Purification of Amplified cDNA Library
				3.1.18 Size-Based Selection of cDNA Libraries
				3.1.19 Ethanol Precipitation of cDNAs
			3.2 Computational Analysis
				3.2.1 Bioinformatic Analysis of CLASH Sequencing Output
				3.2.2 Preparing a Minimum Input File for the Data Analysis
				3.2.3 Removal of Duplicate Hybrids and Folding Unique Chimera
				3.2.4 Comparison of Folding Energies of Experimentally Defined Chimeras and Artificially Generated Control Interactions
				3.2.5 Clustering of Targets with Common Seed Sequences
				3.2.6 Uncovering Enriched Motifs within Seed Sequences
		4 Notes
		References
	Chapter 18: Global Identification of RNA-Binding Proteins in Bacteria
		1 Introduction
		2 Materials
			2.1 Bacterial Culturing
			2.2 Buffers and Reagents
			2.3 Equipment
		3 Methods
			3.1 Verification of Polyadenylation of RNA
			3.2 RNA Harvest and Northern Blotting
			3.3 Determining the Optimal Duration of PAPI Induction
			3.4 UV Cross-Linking and Harvesting of Bacterial Cultures
			3.5 Cell Lysis
			3.6 RNA-Protein Pulldown
			3.7 Recycling of Beads
			3.8 Protein Analysis by SDS-PAGE
		4 Notes
		References
	Chapter 19: An Integrated Affinity Chromatography-Based Approach to Unravel the sRNA Interactome in Nitrogen-Fixing Rhizobia
		1 Introduction
		2 Materials
			2.1 MS2 Aptamer Tagging of the sRNA
			2.2 Culture, Harvest of Bacteria, and Cell Lysis
			2.3 Total RNA Extraction from Bacterial Iysates
			2.4 Affinity Chromatography
			2.5 RT-qPCR Analysis
			2.6 Bioinformatics Analysis
		3 Methods
			3.1 Aptamer Tagging of the sRNAs
			3.2 Cell Growth and Pulse Expression of the MS2-sRNA
			3.3 Quality Check of the Tagging Strategy
				3.3.1 RNA Purification from Harvested Cells
				3.3.2 Assessment of Quality and Quantity of RNA in Samples
			3.4 Affinity Chromatography
			3.5 RNA Purification from the Eluates and Processing for RNAseq
			3.6 Protein Purification from the Eluates and Preparation for Proteomics
			3.7 Data Analysis (RNA-Seq)
			3.8 Data Analysis (Proteomics)
		4 Notes
		References
Part IV: sRNA Structure
	Chapter 20: sRNA Structural Modeling Based on NMR Data
		1 Introduction
		2 Materials
			2.1 Enzymes, Buffers, and Chemicals
			2.2 Equipment
			2.3 Software
		3 Methods
			3.1 RNA In Vitro Transcription for NMR Studies
			3.2 Experimental Determination of sRNA Secondary Structure
			3.3 Structural Modeling of sRNA Using Computational Methods
		4 Notes
		References
	Chapter 21: Circular and Linear Dichroism for the Analysis of Small Noncoding RNA Properties
		1 Introduction
		2 Materials
			2.1 Nucleic Acids, Proteins, Buffers, and Chemicals
			2.2 Materials for Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy
			2.3 Materials for Synchrotron Radiation Linear Dichroism (SRLD) Spectroscopy
		3 Methods
			3.1 Acquisition and Treatment of SRCD Spectra
				3.1.1 Sample Preparation and Loading
				3.1.2 SRCD Data Acquisition
				3.1.3 Spectral Data Treatment
				3.1.4 RNA Spectra Analysis
				3.1.5 Thermal Scans Acquisition
				3.1.6 Specific Case of Proteins Allowing RNA Annealing
			3.2 SRLD Spectra Acquisition and Treatment
				3.2.1 Sample Loading
				3.2.2 SRLD Data Acquisition and Treatment
		4 Notes
		References
Index