piRNA: Methods and Protocols

Preface
Contents
Contributors
Part I: Methods to Study the Nature and Function of piRNAs in Non-classical Organisms
	Chapter 1: Functional Analysis of Individual piRNAs in Aedes aegypti Cells and Embryos Using Antisense Oligonucleotides
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Transfection of AOs in Aag2 Cells
			2.3 Luciferase Reporter Assay
			2.4 RNA Isolation
			2.5 Reverse Transcription and Quantitative  PCR
			2.6 Mosquito Husbandry
			2.7 Injection of Embryos
		3 Methods
			3.1 Design of Antisense Oligonucleotides (AOs)
			3.2 Design of Luciferase Reporter
			3.3 Aag2 Cell Culture
			3.4 Luciferase Reporter Assay
				3.4.1 Transfection of Aag2 Cells
				3.4.2 Luciferase Assay
			3.5 Gene Expression Analysis in Aag2 Cells by RT-qPCR
				3.5.1 Transfection of Aag2 Cells
				3.5.2 RNA Isolation
				3.5.3 cDNA Synthesis and Quantitative  PCR
			3.6 Gene Expression Analysis in Pre-Blastoderm Embryos
				3.6.1 Mosquito Husbandry
				3.6.2 Injection into Pre-Blastoderm Embryos
		4 Notes
		References
	Chapter 2: CRISPR-Mediated Genome Engineering in Aedes aegypti
		1 Introduction
		2 Materials (a List of all Materials)
			2.1 CRISPR Target Site Selection and Validation
			2.2 Injection Mixture Preparation
			2.3 Mosquito Husbandry and Embryonic Microinjection
			2.4 Genetic Crosses, Screening for Mutations and Generation of Homozygous Mutant Lines
		3 Methods
			3.1 CRISPR/Cas9-Mediated Knockout
				3.1.1 Selection and Validation of CRISPR Target Sites
				3.1.2 Preparation of the Injection Mixture
				3.1.3 Mosquito Husbandry and Embryonic Microinjection
				3.1.4 Genetic Crosses, Screening for Mutations, and Generation of Homozygous Mutant Lines
			3.2 CRISPR/Cas9-Mediated Knock-in
				3.2.1 Selection and Validation of CRISPR Target Site
				3.2.2 Preparation of the Injection Mixture
				3.2.3 Mosquito Husbandry and Embryonic Microinjection
				3.2.4 Genetic Crosses, Screening for HDR Insertions, and Generation of Homozygous Mutant Lines
		4 Notes
		References
	Chapter 3: PIWI-Directed DNA Elimination for Tetrahymena Genetics
		1 Introduction
		2 Materials
			2.1 Extraction of Tetrahymena Genomic DNA
			2.2 Construction of coDel-Targeting Plasmid
			2.3 Transformation for Inducing coDel
			2.4 Detecting coDel by Direct PCR
			2.5 Establishing Gene Knockout Cell Lines from Cells with coDel
			2.6 Using coDel to Study DNA Elimination Mechanism
		3 Methods
			3.1 Extraction of Tetrahymena Genomic DNA
			3.2 Construction of coDel-Targeting Plasmid
			3.3 Transformation for Inducing coDel
			3.4 Detecting coDel by Direct PCR
			3.5 Establishing Gene Knockout Cell Lines from Cells with coDel
			3.6 Using coDel to Study DNA Elimination Mechanism
		4 Notes
		References
	Chapter 4: Planarian PIWI-piRNA Interaction Analysis Using Immunoprecipitation and piRNA Sequencing
		1 Introduction
		2 Materials
			2.1 Maintenance of Planarian
			2.2 Preparation of Lysate
			2.3 Immunoprecipitation of Piwi-piRNA Complex
				2.3.1 Using Magnetic  Bead
				2.3.2 Using Sepharose  Bead
			2.4 Immunoprecipitation Quality Assessment (Optional)
			2.5 Purification of Immunoprecipitated  RNA
			2.6 Size Selection of piRNA
			2.7 piRNA Quantity and Quality Assessment
			2.8 piRNA-Seq Library Preparation
			2.9 piRNA-Seq Library Quality Assessment
			2.10 Sequencing
		3 Methods
			3.1 Maintenance of Planarian
			3.2 Preparation of Lysate
			3.3 Immunoprecipitation of Piwi-piRNA Complex
				3.3.1 Using Magnetic  Bead
				3.3.2 With Sepharose  Bead
			3.4 Immunoprecipitation Quality Assessment (Optional)
			3.5 Purification of Immunoprecipitated  RNA
			3.6 Size Selection of piRNA
			3.7 piRNA Quantity and Quality Assessment
			3.8 Small RNA-Seq Library Preparation
			3.9 piRNA-Seq Library Quality Assessment
			3.10 Sequencing
			3.11 Bioinformatic Analysis of Planarian piRNA
		4 Notes
		References
	Chapter 5: Isolation and Processing of Bovine Oocytes for Small RNA Sequencing
		1 Introduction
		2 Materials
			2.1 Bovine Oocyte Collection
			2.2 RNA Extraction
			2.3 β-Oxidation
		3 Methods
			3.1 Bovine Ovary Collection
			3.2 Oocyte Collection
			3.3 GV Stage Oocyte Collection
			3.4 MII Stage Oocyte Collection
			3.5 RNA Extraction
			3.6 β-Oxidation
		4 Notes
		References
	Chapter 6: 3D Imaging and In Situ Hybridization for Uncovering the Functions of MicroRNA in Rice Anther
		1 Introduction
		2 Materials
			2.1 Reagents for In situ Hybridization
			2.2 Reagents for 3D Imaging of Anthers
		3 Methods
			3.1 In Situ Hybridization Using the miR2118 Probe
				3.1.1 Fixation
				3.1.2 Embedding and Sectioning
				3.1.3 Proteinase K Treatment of Samples
				3.1.4 Hybridization
				3.1.5 Post-hybridization (Washing)
				3.1.6 Probe Detection
			3.2 Visualization of the 3D Structure of the Entire Anther
		4 Notes
		References
Part II: Methods to Study Roles of piRNAs in Classic Model Organisms
	Chapter 7: Cloning, Sequencing, and Linkage Analysis of piRNAs
		1 Introduction
		2 Materials
			2.1 Preparation of Total RNA from Fly Ovaries
			2.2 Small RNA Cloning
			2.3 Equipment
			2.4 Software for the Computational Analyses
		3 Methods
			3.1 Preparation of Small RNA Libraries for Illumina Sequencing
				3.1.1 Extracting Total RNA from Fly Ovaries
				3.1.2 2S Ribosomal RNA Depletion
				3.1.3 Radiolabel 19 mer and 35 mer Spike RNA
				3.1.4 Size Selection
				3.1.5 Oxidation of SMALL RNA Pool (Optional)
				3.1.6 3′ Linker Ligation
				3.1.7 5′ Linker Ligation
				3.1.8 Reverse Transcription
				3.1.9 Amplify the Library Using KAPA DNA Polymerase
			3.2 Computational Analysis of Small RNA Sequencing Libraries
				3.2.1 Filter Poor-Quality Reads and Trim Adapter Sequencing
				3.2.2 Filter Reads that Come from Infrastructural RNAs as Well as MicroRNAs
				3.2.3 Map the Reads Against the Genome and Take the Uniquely Mapping Reads
				3.2.4 Map the Reads Against Transposon Reference Sequences
				3.2.5 Evaluating the Complexity of the Sequencing Libraries (Optional)
				3.2.6 Linkage Analysis of Ping-Pong and Phasing piRNA Biogenesis
					Prepare the ``End Counts´´ Table for Transposon-Mapping Reads
					Calculate the Linkage Using R
					Evaluate the Statistical Significance of the Linkage Analysis
				3.2.7 Measure the Downstream Uridine Bias for Phased piRNAs
					Obtain Reads that Are Derived from mRNA 3′ UTRs and Measure the 5′ End Nucleotide Composition
					Measure the Downstream Uridine Bias
		4 Notes
		References
	Chapter 8: Drosophila Genetic Resources for Elucidating piRNA Pathway
		1 Introduction
		2 Materials
			2.1 Drosophila Strains
			2.2 Dissection of Drosophila Gonad
			2.3 Measurement of TE mRNAs
		3 Methods
			3.1 Drosophila Culture and Mating
			3.2 Dissection of Drosophila Gonad
			3.3 Preparation of Total RNAs and RT-qPCR
		4 Notes
		References
	Chapter 9: Generation of Stable Drosophila Ovarian Somatic Cell Lines Using the piggyBac System
		1 Introduction
		2 Materials
			2.1 Preparation of PiggyBac Plasmids
			2.2 OSC Cell Culture
			2.3 Transfection and Single Colony Isolation
		3 Methods
			3.1 PiggyBac Donor Plasmid Design and Cloning Gene of Interest
			3.2 OSC Cell Culture
			3.3 Transfection of Plasmids to OSC
			3.4 Puromycin Selection and Obtaining Monoclonal OSC Line
			3.5 Screening for Positive Clones and Cryopreservation of the Cells
		4 Notes
		References
Part III: Methods to Study Nuclear Regulation by Other Non-coding RNAs
	Chapter 10: Whole-Mount RNA FISH Combined with Immunofluorescence for the Analysis of the Telomeric Ribonucleoproteins in the ...
		1 Introduction
		2 Materials
			2.1 Riboprobe Labeling
			2.2 Tissue Preparation and Fixation
			2.3 Permeabilization
			2.4 RNA FISH
			2.5 Immunostaining
		3 Methods
			3.1 Riboprobe Preparation
			3.2 Preparation and Fixation of Drosophila Ovaries
			3.3 Preparation and Fixation of Drosophila 0-2-h-Old Embryos
			3.4 Pretreatment
				3.4.1 For the Detection of the Nuclear RNA and Proteins at Mid to Late Stages of Oogenesis (See Note 4)
				3.4.2 For the Detection of the Cytoplasmic RNA and Proteins at Mid-Stages of Oogenesis (See Note 6)
				3.4.3 For the Detection of RNA and Proteins at Early Stages of Oogenesis in the Germarium Region (See Also the Protocol)
				3.4.4 For the Detection of RNA and Proteins in 0- to 2-h-Old Drosophila Embryos
			3.5 RNA FISH
			3.6 RNA Detection and Immunostaining
		4 Notes
		References
	Chapter 11: CRISPR-Mediated Activation of Transposable Elements in Embryonic Stem Cells
		1 Introduction
		2 Materials
			2.1 Cell Lines
			2.2 Reagents
				2.2.1 Plasmids (See Note 1)
				2.2.2 Media for Cell Culture and Passaging
				2.2.3 Other Reagents
			2.3 Equipment
			2.4 Software
			2.5 Reagent Setup
		3 Methods
			3.1 Generation and Validation of CRISPRa Transgenic ESCs
			3.2 Design of RLTR10B2-Targeting gRNA and Construction of a gRNA-Expressing Lentiviral Vector
				3.2.1 Design the gRNA Oligos and Clone Them into the pSpCas9(BB)-2A-Puro (pX459) V2.0 Plasmid
				3.2.2 Assembly of 5xRLTR10B2-Targeting gRNA Array and Construction of Lentiviral gRNA Expression Vector
			3.3 Production of Lentiviral Particles, Harbor 5x RLTR10B2-Targeting gRNA Array by Transfecting HEK293T
			3.4 Generation and Validation of RLTR10B2-Targeting CRISPRa ESCs
			3.5 Functional Evaluation of CRISPRa-Mediated Activation of Spermatogenic ERV Enhancers: RLTR10B2 in ESCs
			3.6 RNA-seq Data Processing and Analysis
		4 Notes
		References
	Chapter 12: Method for Evaluating Effects of Non-coding RNAs on Nucleosome Stability
		1 Introduction
		2 Materials
			2.1 Transcription of RNA In Vitro
			2.2 Nucleosome Reconstitution and Purification
			2.3 Thermal Stability Assay of Nucleosomes in the Presence of RNAs
			2.4 General Equipment
			2.5 Buffers
		3 Methods
			3.1 In Vitro RNA Transcription
			3.2 Nucleosome Preparation for Thermal Stability Assay
				3.2.1 Assembly of Histone H2A-H2B Dimer and H3-H4 Tetramer
				3.2.2 Purification of H2A-H2B Dimer and H3-H4 Tetramer by Size Exclusion Chromatography
				3.2.3 Nucleosome Reconstitution from Purified Histone Complexes
					Reconstitution of the Nucleosome
						Small-Scale Reconstitution
						Large-Scale Reconstitution
					Purification of Nucleosomes
			3.3 Thermal Stability Assay of the Nucleosome in the Presence of Eleanor2 RNA
			3.4 Analysis of the Disrupted Nucleosomes by Native-PAGE
			3.5 Thermal Stability Assay of the Nucleosome in the Presence of Other RNAs
		4 Notes
		References
	Chapter 13: Revisiting the Glass Treatment for Single-Molecule Analysis of ncRNA Function
		1 Introduction
		2 Materials
			2.1 General Buffers, Reagents, and Cells
			2.2 TIRF Microscope Setup
			2.3 HaloTag and SNAP-Tag Ligand Having Both Cy5 and Biotin
			2.4 Dicer-2 Protein Labeling with HaloTag Ligands
			2.5 Preparation of Fluorescently Labeled dsRNA Target
			2.6 Glass Cleaning and Passivation
			2.7 Preparation of the Observation Chamber
			2.8 Software
		3 Methods
			3.1 Dicer-2 Protein Labeling with HaloTag Ligands
				3.1.1 HaloTag Cy5-Biotin Preparation (See Note 2)
				3.1.2 Halo-Dicer-2 Expression
				3.1.3 Halo-Dicer-2 Labeling
			3.2 Preparation of Fluorescently Labeled dsRNA Targets
				3.2.1 Transcription and Annealing
			3.3 Preparation of the PEG-Coated Quartz Slides and Coverslips (Method #1, Fig. 2,)
				3.3.1 Pre-cleaning of the Quartz Slides and Coverslips
				3.3.2 Amino-silanization of Slides and Coverslips
				3.3.3 Surface Passivation Using Polymer
			3.4 Preparation of the PEG-Coated Quartz Slides and Coverslips (Method #2,) (See Note 4)
				3.4.1 Pre-cleaning of the Quartz Slides and Coverslips
				3.4.2 Amino-silanization of Slides and Coverslips
				3.4.3 Surface Passivation Using Polymer
			3.5 Preparation of the PEG-Coated Quartz Slides and Coverslips (Method #3,) (See Note 5)
				3.5.1 Pre-cleaning of the Quartz Slides and Coverslips
				3.5.2 Amino-silanization of Slides and Coverslips
				3.5.3 Surface Passivation Using Polymer
			3.6 Preparation of the PEG-Coated Quartz Slides and Coverslips (Method #4) (See Note 6)
				3.6.1 Pre-cleaning of the Quartz Slides and Coverslips
				3.6.2 Amino-silanization of Slides and Coverslips
				3.6.3 Surface Passivation Using Polymer
			3.7 Clean and Reuse of Quartz Slides
			3.8 Single-Molecule Imaging (Fig. 2) (See Note 7)
				3.8.1 Preparation of Single-Molecule Observation Chamber
				3.8.2 Continuous Monitoring of Spot Appearance
				3.8.3 Data Processing and Image Analysis
		4 Notes
		References
	Chapter 14: Low Input Genome-Wide DNA Methylation Analysis with Minimal Library Amplification
		1 Introduction
		2 Materials
			2.1 Commercial Kits, Enzymes, and Consumables
			2.2 Stock Solutions
			2.3 Working Solutions
			2.4 Adaptors and Primers (100 μM, Oligonucleotide Purification Column Grade)
			2.5 Equipment
		3 Methods
			3.1 Cell Disruption
			3.2 Bisulfite Conversion
			3.3 Adaptor Tagging/First Strand Synthesis
			3.4 DNA Purification
			3.5 Complexation of Biotinylated DNA with Streptavidin Beads
			3.6 Adaptor Tagging/Second Strand Synthesis
			3.7 Chase Reaction
			3.8 Elution and Extension of Template DNA
			3.9 DNA Purification
			3.10 Library Amplification
			3.11 DNA Purification
			3.12 Library Quantification
			3.13 Library Dilution for NovaSeq Sequencing (SP and S1 Flow Cell, Xp Workflow)
			3.14 Bioinformatic Analysis of WGBS Data
		4 Notes
		References
	Chapter 15: Solid-Support Directional (SSD) RNA-Seq as a Companion Method to CLIP-Seq
		1 Introduction
		2 Materials and Equipment
			2.1 Isolation of Total RNA from a Tissue or Cell Sample Using TRIzol
			2.2 Ribosomal RNA Depletion Using RiboMinus Eukaryote Kit for RNA-Seq
			2.3 Fragmentation of rRNA-Depleted Total RNA
			2.4 Biotinylated Adapter Ligation and Capture of Ligated RNAs Using M280 Streptavidin Dynabeads
			2.5 On-Beads Ligation with the 5′ Adapter
			2.6 Reverse Transcription and PCR Amplification of Adapter-Ligated RNA
		3 Methods
			3.1 Isolation of Total RNA from a Tissue or Cell Sample Using TRIzol
			3.2 Ribosomal RNA Depletion Using RiboMinus Eukaryote Kit for RNA-Seq
			3.3 Fragmentation of rRNA-Depleted Total RNA and Dephosphorylation
			3.4 Biotinylated Adapter Ligation and Capture of Ligated RNAs Using M280 Streptavidin Dynabeads
			3.5 On-Beads Ligation with the 5′ Adapter
			3.6 Reverse Transcription and PCR Amplification of Adapter-Ligated RNA
			3.7 Pre-processing of Sequencing Data and Mapping
		4 Notes
		References
	Chapter 16: UPA-Seq-Based Search Method for Functional lncRNA Candidates
		1 Introduction
		2 Materials
			2.1 Equipment for Sample Preparation
				2.1.1 Equipment for Sample Preparation (Common)
				2.1.2 Equipment for Sample Preparation (Cultured Cell Sample)
				2.1.3 Equipment for Sample Preparation (Tissue Sample)
			2.2 Stock Solutions
				2.2.1 Stock Solutions (Common)
				2.2.2 Stock Solution (Cultured Cell Sample)
			2.3 Working Solutions
				2.3.1 Working Solution (Common)
				2.3.2 Working Solutions (Cultured Cell Sample)
			2.4 Other Reagents
		3 Methods
			3.1 Preparation of Samples
				3.1.1 Preparation of Samples from Cultured Cells and UV Irradiation
				3.1.2 Preparation of Samples from Tissues and UV Irradiation
			3.2 RNA Extraction
			3.3 RNA Sequencing (RNA-seq)
			3.4 Analysis of RNA Sequence Data
		4 Notes
		References
	Chapter 17: Large-Scale Analysis of RNA-Protein Interactions for Functional RNA Motif Discovery Using FOREST
		1 Introduction
		2 Materials
			2.1 RNA Structure Library Design
			2.2 In Vitro Transcription of RNA Probes and the RNA Structure Libraries
			2.3 3′-Terminal Cy5 Labeling
			2.4 Target RNA Enrichment
			2.5 Hybridization and Microarray Scanning
		3 Methods
			3.1 RNA Structure Library Design
			3.2 In Vitro Transcription of the RNA Structure Library
			3.3 3′-Terminal Cy5 Labeling
			3.4 Target RNA Enrichment
				3.4.1 RNA Pull-Down with His-Tagged Proteins
				3.4.2 RNA Immunoprecipitation with Flag-Tagged Proteins
			3.5 Hybridization and Microarray Scanning
			3.6 Calculation of Binding Intensities
		4 Notes
		References
Part IV: Bioinformatic and Biophysical Methods to Study Non-coding RNAs
	Chapter 18: Computational Methods for the Discovery and Annotation of Viral Integrations
		1 Introduction
		2 Materials
			2.1 Required Programs and Scripts
				2.1.1 Procedure 1, Annotation of EVEs in a Genome Assembly
				2.1.2 Procedure 2, Assessment of EVEs Polymorphism
				2.1.3 Procedure 3, Identification of Novel EVEs
			2.2 Computational Power
			2.3 Notes Before Beginning
		3 Methods
			3.1 Annotation of EVEs in a Genome Assembly
				3.1.1 Preliminary Operations
				3.1.2 Running the Pipeline
				3.1.3 Pipeline Output
			3.2 Assessment of EVE Polymorphism
				3.2.1 Preliminary Operations
				3.2.2 Running the SVD Pipeline
				3.2.3 SVD Pipeline Output
			3.3 Identification of Novel EVEs
				3.3.1 Preliminary Operations
				3.3.2 Running Vy-PER
				3.3.3 Vy-PER Output
				3.3.4 Running ViR Module 1
				3.3.5 ViR Module 1 Output
				3.3.6 Running ViR_LTFinder.sh
				3.3.7 ViR_LTFinder.sh Output
		4 Notes
		References
	Chapter 19: Bioinformatics Approaches for Determining the Functional Impact of Repetitive Elements on Non-coding RNAs
		1 Introduction
		2 Emerging Roles of Repetitive Elements in ncRNAs
			2.1 Nuclear Retention
			2.2 Biomolecular Condensates
			2.3 Gene Expression
			2.4 RNA Processing and Translation
		3 Bioinformatics Approaches for Studying the Role of Repetitive Elements in ncRNAs
			3.1 De Novo Identification of Repetitive Elements
			3.2 Tissue/Tumor-Specific Expression
			3.3 Subcellular Localization
			3.4 ncRNA-RNA Interactions
			3.5 ncRNA-DNA Interactions
			3.6 ncRNA-Protein Interactions
		4 Concluding Remarks
		References
	Chapter 20: Extending and Running the Mosquito Small RNA Genomics Resource Pipeline
		1 Introduction
		2 Materials
			2.1 Installation and Dependencies
			2.2 Reference and Supporting Files
			2.3 Sample Naming Convention for Input Files and Computing Cluster Space Considerations
		3 Methods
			3.1 The Gene-Centric Script, a Central Initial Processing  Step
			3.2 miRNA Counting and Other Read-Lengths Separation Scripts
			3.3 Transposable Elements, Viruses, Structural RNAs, and Wolbachia Genomes (TVSW) Scripts
			3.4 Saving Results to Sharing Folder
			3.5 piRNA Ping-Pong and Phasing Patterns Analysis
		4 Notes
			4.1 Noting the MSRG Input Parameter for the 3′ Adapter Sequence in the Small RNA Libraries.
			4.2 Visualizing WIG/BigWig Plots on the Integrate Genome Viewer (IGV) Browser Requires Matched Set GTF File and Genome  File
			4.3 Why Are the Gene-Centric Script and Phasing Scripts So  Slow?
		References
	Chapter 21: Preparation of Non-overlapping Transposable Elements (TEs) Annotation by Interval Tree
		1 Introduction
		2 Materials
			2.1 Computer
			2.2 Computational Environment
		3 Method
			3.1 Preparation of Repeat Annotation
			3.2 Remove Overlapping Regions
				3.2.1 Principle
				3.2.2 Usage
			3.3 Output Files
			3.4 Options
			3.5 Further Filtering
		4 Notes
			4.1 Repeat Library
		References
	Chapter 22: Statistical Thermodynamics Approach for Intracellular Phase Separation
		1 Introduction
			1.1 Phase Separation and Biomolecular Condensates
			1.2 RNA and Phase Separation
		2 Materials
		3 Methods
			3.1 Basic Principle of Phase Separation
			3.2 Phase Separation in Polymer Solution
				3.2.1 Flory-Huggins Theory
				3.2.2 Excluded Volume Interactions Between Monomers in Polymers
			3.3 Phase Separation and Self-Assembly
			3.4 Micellization of Block Copolymers
				3.4.1 Free Energy of a Block Copolymer Micelle
				3.4.2 Free Energy of a Block Copolymer Solution
			3.5 Summary and Extension to Intracellular Phase Separation Research
			3.6 Example of Experimental Validations of Theoretical Predictions
		4 Notes
		References
Index