Circular RNAs

Preface
	References
Contents
Contributors
Part I: circRNA Purification
	Chapter 1: Purification of Circular RNAs Using Poly(A) Tailing Followed by RNase R Digestion
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Total RNA Extraction and Confirmation of RNA Quality
			2.3 Poly(A) Tailing
			2.4 RNase R Digestion
			2.5 RT-qPCR
			2.6 RNA-seq Library Preparation and Illumina Sequencing
			2.7 RNA-seq Data Analysis
		3 Methods
			3.1 Culture and Extraction of Total RNA from HeLa Cells
			3.2 Poly(A) Tailing of Total  RNA
			3.3 RNase R Digestion to Remove Linear  RNAs
			3.4 RT-qPCR to Examine Individual Transcript Levels
			3.5 RNA-seq Library Preparation and Illumina Sequencing
			3.6 Annotation of Circular RNAs from RNA-seq Data Using CIRI2
			3.7 Annotation of Circular RNAs from RNA-seq Data Using CIRCexplorer2
			3.8 Generate Genome Browser Tracks for Visualization of Circular  RNAs
		4 Notes
		References
Part II: In Silico circRNA Profiling
	Chapter 2: State-of-the-Art Circular RNA Analytics Using the Circtools Software Suite
		1 Introduction
		2 Materials
			2.1 Required Hardware
			2.2 Required Software
			2.3 Required Data
			2.4 General Notes
			2.5 How to Get Support
			2.6 Download and Preparation of Raw Sequencing Data
		3 Methods
		4 Conclusion
		References
	Chapter 3: Exploring Circular RNA Profile and Expression in Extracellular Vesicles
		1 Introduction
		2 Materials
			2.1 Biofluids Separation and Storage
			2.2 Purification of EVs
			2.3 EV RNA Isolation
			2.4 Library Preparation for RNA-Seq
			2.5 Hardware and Environment for EV-circRNA Analysis
			2.6 Software and Environment for EV-circRNA Analysis
		3 Method
			3.1 Biofluids Separation and Storage
			3.2 Purification of EVs
			3.3 EV-RNA Isolation
			3.4 Library Preparation for RNA-Seq
			3.5 Quality Control for Sequencing Data
			3.6 EV CricRNA Detection with CIRI2
			3.7 EV CricRNA Detection with ASJA
			3.8 Characterization of EV-CircRNA Profile
		4 Notes
		References
Part III: circRNA Detection, Sequence Validation and Quantification
	Chapter 4: In Situ Hybridization of circRNAs in Cells and Tissues through BaseScope Strategy
		1 Introduction
		2 Materials
			2.1 Probe Design
			2.2 Reagents
			2.3 Solutions
			2.4 Cell Culture
				2.4.1 Coating Solutions
				2.4.2 Cell Culture Reagents
				2.4.3 Cells
				2.4.4 Cell Culture Medium
			2.5 Equipment
		3 Methods
			3.1 Culturing and Fixation Protocols for Neuronal Cells
				3.1.1 Murine Neuroblastoma Cell Line (N2a) (Fig. 2a)
				3.1.2 Mouse Embryonic Stem Cells (mESC) Differentiation into Spinal Motor Neurons
				3.1.3 Motor Neurons (MNs) Seeding and Fixation (Fig. 2b)
			3.2 Fixation and Embedding Protocol for Brain Tissues
			3.3 Sample Pretreatment
				3.3.1 N2a and MNs
				3.3.2 Tissues (Fig. 4)
			3.4 ZZ-Probes Hybridization
			3.5 BaseScope v2 Amplifiers
			3.6 Signal Detection
			3.7 Perform Immunofluorescence (IF) After BaseScope Hybridization
			3.8 DAPI Detection
			3.9 Mounting IBIDI/Tissues or Glass Coverslips
			3.10 Acquisition at Confocal Microscope (Fig. 2)
			3.11 Image Analysis
			3.12 Structured Illumination Microscopy (SIM) (Fig. 5)
		4 Notes
		References
	Chapter 5: Sanger Sequencing to Determine the Full-Length Sequence of Circular RNAs
		1 Introduction
		2 Materials
			2.1 Retrieve CircRNA Mature Sequence and Primer Designing
			2.2 cDNA Synthesis and RT-PCR
			2.3 Agarose Gel Electrophoresis and Sanger Sequencing
		3 Method
			3.1 CircRNA Backsplice Junction Validation
				3.1.1 Retrieve CircRNA Sequence
				3.1.2 Divergent Primer Preparation
				3.1.3 Reverse Transcription and PCR Amplification of CircRNA  BSJ
				3.1.4 Gel Electrophoresis to Visualize PCR Product and Sanger Sequencing to Validate the Backsplice Junction Sequence
			3.2 Identification of CircRNA Splice Variants Through CircRNA PCR Using Full-Length Primers
				3.2.1 Full-Length CircRNA Primer Design for Full-Length PCR Amplification of Target CircRNAs and Their Splice Variants
				3.2.2 Depletion of Linear RNAs with RNase R Treatment of Total RNA for the Enrichment of circRNAs
				3.2.3 Full-Length cDNA of CircRNA by Rolling Circle Reverse Transcription
				3.2.4 CircRNA-Rolling Circle Amplification (CircRNA-RCA) Using Full-Length Primers
				3.2.5 Full-Length CircRNA Sequence Identification by Sanger Sequencing
		4 Notes
		References
	Chapter 6: Droplet Digital PCR for the Detection and Quantification of Bona Fide CircRNAs
		1 Introduction
		2 Materials
			2.1 Primer Design
			2.2 RNase R Treatment, RT Reagents Equipments
			2.3 ddPCR Reagents
			2.4 ddPCR Equipment
			2.5 ddPCR Instruments
			2.6 ddPCR Data Analysis
		3 Methods
			3.1 Divergent Primers Design
			3.2 RNase R Treatment and cDNA Synthesis
			3.3 cDNA and Primer Mix Preparation
			3.4 ddPCR Mix Assembly Using EvaGreen-Based Assay
			3.5 ddPCR Droplet Generation and Transfer to PCR Plate
				3.5.1 Manual Droplet Generation and Transfer
				3.5.2 Automated Droplet Generation and Transfer
			3.6 PCR for EvaGreen-Based ddPCR Assays
			3.7 Droplets Reading
			3.8 ddPCR Data Analysis
		4 Notes
		References
	Chapter 7: Targeted Sequencing of Circular RNAs for Illumina-Based Counting and Nanopore Structure Determination
		1 Introduction
		2 Materials
			2.1 Lexogen Library Generation and Amplification
			2.2 Illumina Sequencing
			2.3 Nanopore cDNA Sequencing
			2.4 Hardware Requirements
			2.5 Software Dependencies and Installation
		3 Methods
			3.1 Design of First Strand Synthesis Primers
			3.2 Library Generation
			3.3 Amplification of Libraries
			3.4 Nanopore cDNA Sequencing
			3.5 Illumina Read Processing
			3.6 Nanopore Read Processing
		4 Results
			4.1 Library and Sequencing Quality Control
			4.2 Analysis of circRNA Levels by Illumina Sequencing
			4.3 Identification of Full-Length circRNA Architecture by Nanopore Long-Read Sequencing
		5 Notes
		References
	Chapter 8: Nanopore-Mediated Sequencing of Circular RNA
		1 Introduction
		2 Material
			2.1 Enriching CircRNAs from Total  RNAs
			2.2 Long-Read Sequencing of Enriched CircRNA  Pool
			2.3 Full-Length Sequencing of a Targeted Panel of CircRNAs
		3 Methods
			3.1 Enriching CircRNA from Total RNA (Outlined in Fig. 1a) (See Note 1)
			3.2 Long-Read Sequencing of Enriched CircRNA Pool (circNick-LRS Protocol; Outlined in Fig. 1b) (See Note 5)
			3.3 Full-Length Sequencing of a Targeted Panel of CircRNAs (circPanel-LRS)
				3.3.1 CircRNA Specific Primer (CSP) Design
				3.3.2 Full-Length CircRNA Sequencing
		4 Notes
		References
Part IV: Functional Study: Tools for Loss- and Gain-of Function Analysis
	Chapter 9: In Vivo Tissue-Specific Knockdown of circRNAs Using shRNAs in Drosophila melanogaster
		1 Introduction
		2 Materials
			2.1 Design shRNA and 5´/3´ Shifts
			2.2 Cloning of shRNA
			2.3 Expression of the shRNA
			2.4 qPCR
			2.5 Identify Transcripts with Long Complementary Stretches to the shRNA
			2.6 3´UTR Sequence Enrichment Analysis
		3 Methods
			3.1 Design of shRNAs and 5´/3´ Shifts
			3.2 Cloning the shRNA for circRNA Knockdown in Drosophila
			3.3 Expression of the shRNA by Crossing the Transgenic UAS-shRNAs with GAL4-Drivers
			3.4 Verify the Specificity of the shRNA Knockdown of circRNAs by  qPCR
			3.5 Identify Putative Off-Targets Due to Long Stretches of Complementarity and Evaluate  Them
			3.6 Evaluate Putative AGO1 Off-Target Effects Using Sequence Enrichment Analysis in the UTR of Deregulated Genes
		4 Notes
		References
	Chapter 10: The Functional Circular RNA Screening via RfxCas13d/BSJ-gRNA System
		1 Introduction
		2 Materials
			2.1 Biological Materials
			2.2 Reagents
			2.3 Equipment
			2.4 Buffer Preparations
			2.5 Oligos
		3 Methods
			3.1 Construction of the BSJ-gRNA Library
			3.2 Quality Evaluation of the Assembled BSJ-gRNA Library
			3.3 gRNA Library Lentivirus Packaging and Titer
			3.4 BSJ-gRNA Library Infection and Functional circRNA Screening
			3.5 Next-Generation Sequencing (NGS) Library Preparation and Deep Sequencing
			3.6 Data Analysis of Screening Results via CDCscreen
			3.7 Validation of circRNA Candidates Identified by High-Throughput Screening
			3.8 Results
		4 Notes
		References
	Chapter 11: Base-Editor-Mediated circRNA Knockout by Targeting Predominantly Back-Splice Sites
		1 Introduction
		2 Materials
			2.1 Hardware and Software
			2.2 Reference Genome and Gene Annotation
			2.3 RNA-Seq Datasets
			2.4 Cell Lines and Agents for Evaluating Knockout Effect
		3 Methods
			3.1 Genome-Wide Profiling of circRNAs
			3.2 Selection of Predominantly Circularized Exons for Base Editing
			3.3 Evaluation of Knockout Effect
				3.3.1 Plasmid Construction
				3.3.2 Cell Culture and Transfection
				3.3.3 Calculation of Base Editing Ratio
				3.3.4 RNA Isolation and Detection
		4 Discussion
		5 Notes
		References
	Chapter 12: Directed Circularization of a Short RNA
		1 Introduction
			1.1 Biological Relevance of Circular  RNA
			1.2 Common Circularization Strategies
		2 Materials
			2.1 DNA Analysis
			2.2 DNA-Dependent In Vitro Transcription of  RNA
			2.3 RNA Analysis
				2.3.1 Polyacrylamide  Gels
				2.3.2 Reverse Transcription  PCR
		3 Methods
			3.1 Klenow Reaction
			3.2 In Vitro Transcription with GMP Priming and RNA Isolation
			3.3 RNA Purification by Precipitation
			3.4 Ribozyme Reaction
			3.5 Reverse Transcription  PCR
		4 Results
		5 Notes
			5.1 GMP Priming
			5.2 Polyacrylamide Gel Electrophoresis
			5.3 Ribozyme Reaction
		References
	Chapter 13: Engineering Synthetic circRNAs for Efficient CNS Expression
		1 Introduction
		2 Materials
		3 Methods
			3.1 Efficient Backsplicing CircRNA
				3.1.1 CircRNA Cassette Design
				3.1.2 Synthesizing Fragments for Cassette
				3.1.3 Linearization of Backbone
				3.1.4 Gibson Assembly of Backbone and Inserts
				3.1.5 Transformation
			3.2 Cell Culture
				3.2.1 Cell Maintenance and Preparation
				3.2.2 Transfection of Adherent HEK293 Cells
			3.3 RNA Extraction
			3.4 RNase R Treatment
			3.5 RT-PCR and qPCR
			3.6 Recombinant AAV Vector Production
				3.6.1 Insertion of circRNA Cassette Between  ITRs
				3.6.2 Upstream AAV Production
				3.6.3 Determination of Vector Genome Titers
			3.7 Intracerebroventricular (ICV) Injections of AAV-circRNA Vectors into  Mice
				3.7.1 Administration of AAV-circRNA Vectors
				3.7.2 Harvesting Tissue
				3.7.3 Preparation of Tissue
				3.7.4 Immunohistochemistry
				3.7.5 Histological Quantification of Vector Performance
		4 Notes
		References
	Chapter 14: Encapsulating In Vitro Transcribed circRNA into Lipid Nanoparticles Via Microfluidic Mixing
		1 Introduction
			1.1 Basic Aspects of Circular RNA Biology
			1.2 Translational Potential of Circular RNAs
			1.3 Circular RNAs for Clinical Use as a Therapeutic Intervention
			1.4 Lipid Nanoparticle (LNP)-Based Delivery of RNA Therapeutics
		2 Materials
			2.1 LNP Packaging
				2.1.1 Pre-formulation Preparation
				2.1.2 LNP Formulation
			2.2 Downstream Processing and Particle Characterization
				2.2.1 Dialysis, Concentration, and Sterile Filtration
				2.2.2 RiboGreen Assay to Quantify Encapsulation Efficiency
				2.2.3 Particle Sizing and PDI Via Dynamic Light Scattering
		3 Methods
			3.1 In Vitro Transcribed Circular RNA LNP Packaging
				3.1.1 Pre-formulation Preparation and RNA Dilution
				3.1.2 LNP Packaging-Commercial
				3.1.3 Downstream Processing-Dialysis, Concentration, and Sterilization
				3.1.4 Particle Characterization
			3.2 Transfection
				3.2.1 Transfection of In Vitro Transcribed Circular RNA in Cardiomyocytes
				3.2.2 LNP-Based Transfection
			3.3 Pre-clinical Application of IVT Circular RNA in NRCMs to Gain Cardioprotection
		4 Notes
		References
Part V: Functional Study: Biogenesis and Translation
	Chapter 15: Characterizing Post-transcriptional Modifications of circRNAs to Investigate Biogenesis and Translation
		1 Introduction
		2 Materials
			2.1 circRNA Isolation
			2.2 m6A Immunoprecipitation
				2.2.1 MeRIP-seq
				2.2.2 miCLIP
			2.3 Peak Calling
				2.3.1 MeRIP-seq
				2.3.2 miCLIP
			2.4 Validation of m6A Presence on Specific Targets
			2.5 Effects of m6A Factors Downregulation on circRNAs
				2.5.1 Writers/Readers/Erasers Downregulation
				2.5.2 Assessing circRNA Level Upon METTL3 Knockdown
			2.6 Identifying Which Step of RNA Metabolism Is Affected Upon Inhibition of RNA Modification
				2.6.1 Effects on circRNA Localization
				2.6.2 Effects on circRNA Decay
				2.6.3 Effects on circRNA Translation
			2.7 RNA Modifications Site-Specific Mutagenesis
			2.8 Assessing Writers/Readers/Erasers Binding on circRNAs
		3 Methods
			3.1 circRNA Isolation
			3.2 m6A Immunoprecipitation
				3.2.1 MeRIP-seq
				3.2.2 miCLIP
			3.3 Peak Calling
				3.3.1 MeRIP-seq
				3.3.2 miCLIP
			3.4 Validation of m6A Presence on Specific Targets
			3.5 Effects of m6A Factors Downregulation on circRNAs
				3.5.1 Writers/Readers/Erasers Downregulation
				3.5.2 Assessing circRNA Level Upon METTL3 Knockdown
			3.6 Identifying Which Step of RNA Metabolism Is Affected Upon Inhibition of RNA Modification
				3.6.1 Effects on circRNA Localization
				3.6.2 Effects on circRNA Decay
				3.6.3 Effects on circRNA Translation
			3.7 RNA Modifications Site-Specific Mutagenesis
			3.8 Assessing Writers/Readers/Erasers Binding on circRNAs
		4 Notes
		References
Part VI: circRNA Mechanisms of Action
	Chapter 16: Native Circular RNA Pulldown Method to Simultaneously Profile RNA and Protein Interactions
		1 Introduction
		2 Materials
			2.1 Tissue Culture and Transfection
			2.2 Native Co-precipitation (circRNA-RNA and circRNA-Protein Pulldown)
			2.3 Profiling RNA Interactions
				2.3.1 Validation of circRNA Pulldown
			2.4 Profiling Protein Interactions
		3 Methods
			3.1 Design of circRNA Pulldown Oligonucleotide Probe
				3.1.1 Probe Design
				3.1.2 Overexpression of circRNA (Optional)
				3.1.3 Tissue Culture and Transfection
			3.2 Native Co-precipitation (circRNA-RNA and circRNA-Protein Pulldown)
			3.3 Profiling RNA Interactions
				3.3.1 Elution and Purification of RNA from Beads
				3.3.2 Validation of circRNA Pulldown with qRT-PCR
			3.4 Profiling Protein Interactions
		4 Notes
		References
	Chapter 17: Screening and Characterization of Functional circRNAs in Neuronal Cultures
		1 Introduction
		2 Materials
			2.1 Hardware
			2.2 Software
			2.3 Online Resources
			2.4 Reagents
				2.4.1 Cell Culture and Stimulation
				2.4.2 RNA Extraction and Preparation
				2.4.3 RNAi Approaches
				2.4.4 Transfection
				2.4.5 Immunocytochemistry
		3 Methods
			3.1 circRNA Candidate Discovery: Compartment and Activity-Dependent Enrichment of circRNAs
			3.2 Screening Criteria for circRNA Screen Candidates
				3.2.1 Filtering of Process-Enriched circRNAs
			3.3 Considerations for RNAi Against circRNA
				3.3.1 siRNA Design
				3.3.2 circRNA Knockdown Validation with siRNAs/shRNAs
			3.4 RNAi Functional Assay for Synapse Density
			3.5 Characterization of circRNA Function by Integrative ``Omics´´ Approach
				3.5.1 Poly-A Sequencing of circRNA Knockdown
				3.5.2 EnrichMir Web Tool miRNA Target Enrichment Analysis
				3.5.3 ScanMir Web Tool: Versatile microRNA Target Prediction for circRNAs
		4 Notes
		References
Index