Transposable Elements: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: An Overview of Best Practices for Transposable Element Identification, Classification, and Annotation in Eukaryotic...
1 Introduction
2 Materials
2.1 Databases
2.2 Software
2.3 REPET Package Dependencies
3 Methods
3.1 TEdenovo Detection
3.2 TE Classification
3.3 TE Annotation
3.4 Post-processing for RepeatMasker Outputs
3.5 Repeat Divergence Landscapes
4 Notes
References
Chapter 2: Assembly-Free Detection and Quantification of Transposable Elements with dnaPipeTE
1 Introduction
2 Materials
2.1 Computing Resources
2.2 Additional Programs
2.3 dnaPipeTE Installation
3 Methods
3.1 Pre-processing
3.1.1 Filtering out Nonhost DNA
3.1.2 mtDNA Reads
3.2 Standard dnaPipeTE Command
3.3 Coverage Calibration
3.4 Comparative Analysis with dnaPipeTE
3.4.1 Run dnaPipeTE with Each Species
3.4.2 Classify the Repeats and Rerun Quantification
3.4.3 dnaPipeTE Outputs and Post-processing
3.4.4 Comparing the Proportion of Shared TE Families
4 Notes
References
Chapter 3: Detecting Horizontal Transfer of Transposons
1 Introduction
2 Materials
2.1 Software List
3 Methods
3.1 Workflow A: Ab Initio
3.2 Workflow B: Global HTT Screening of Specific TEs
3.3 Workflow C: HTT Candidate Validation
4 Notes
References
Chapter 4: Genotyping of Transposable Element Insertions Segregating in Human Populations Using Short-Read Realignments
1 Introduction
1.1 Relevance of TE Insertions in Human Genomics
1.2 Characteristics of Human TE Insertion Polymorphisms
1.3 Bioinformatic Detection and Genotyping of TE Insertion Polymorphisms
1.4 Protocol Overview
2 Materials
2.1 High-Performance Computing (HPC) System
2.2 MELT2, ERVcaller, and TypeREF Installation
2.3 Dependent Bioinformatic Tool Installation
2.4 Human/TE Reference Sequence and Annotation Files
2.5 Samples, Sequencing Data, and Initial Analyses
3 Methods
3.1 Genotype Non-reference TE Insertions
3.1.1 Use MELT2 to Genotype Non-reference TE Insertions
3.1.2 Use ERVcaller to Detect and Genotype Non-reference TE Insertions
3.2 Genotype Reference TE Insertions
3.2.1 Discovery of Polymorphic Reference TE Insertions and Initial Genotyping with MELT2
3.2.2 Refining Reference Alu Genotypes with TypeREF
4 Notes
References
Chapter 5: A Pangenome Approach to Detect and Genotype TE Insertion Polymorphisms
1 Introduction
2 Materials
2.1 Hardware
2.2 Software
2.3 Data
3 Methods
3.1 Building the TE Pangenome
3.2 Aligning and Processing Reads
3.3 Genotyping Variants
4 Notes
References
Chapter 6: Experimental Validation of Transposable Element Insertions Using the Polymerase Chain Reaction (PCR)
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers
2.3 Equipment
3 Methods
3.1 Primer Design (Timing: 30 min)
3.1.1 Get the Nucleotide Sequence of the Region Where the TE Is Annotated
3.1.2 Obtain the Primer Sequences from the Sequence Region of the TE
3.1.3 Positive Control
3.1.4 Buy Primers
3.2 Genomic DNA Extraction (Timing: 1 h)
3.3 PCR Reaction (Timing: 2.5 h Approximately - Depending on the PCR Program)
3.4 Electrophoresis (Timing: 1 h)
3.5 Result Interpretation and Expected Outcomes
4 Notes
References
Chapter 7: Quantification of LINE-1 RNA Expression from Bulk RNA-seq Using L1EM
1 Introduction
2 Materials
3 Methods
3.1 Software Installation and Setup
3.2 Execution
3.3 Output
3.4 Feeding L1EM Results Forward to Differential Expression
4 Notes
References
Chapter 8: Genome-Wide Young L1 Methylation Profiling by bs-ATLAS-seq
1 Introduction
2 Materials
2.1 Instruments and Small Equipment
2.2 Oligodeoxynucleotides (ODN)
2.3 DNA Extraction
2.4 DNA Shearing
2.5 End-Repair, A-Tailing, and Adapter Ligation
2.6 Bisulfite Conversion
2.7 Suppression PCR
2.8 Library Quantification
2.9 Sequencing
2.10 Software
3 Methods
3.1 DNA Extraction from Cultured Cells
3.1.1 DNA Extraction
3.1.2 Genomic DNA Quality Control and Quantification
3.2 DNA Shearing
3.2.1 Preparation
3.2.2 Shearing Procedure
3.2.3 Concentrate the Sonicated DNA and Measure Fragment Size
3.3 End-Repair, A-Tailing, and Adapter Ligation
3.3.1 End-Repair
3.3.2 A-Tailing
3.3.3 Adapter Ligation
3.4 Bisulfite Conversion
3.4.1 Desulfonization and Cleanup
3.5 Suppression PCR
3.6 Double-Size Selection
3.7 Quality Control and Quantification of Libraries
3.8 Illumina Sequencing
3.8.1 Multiplexing
3.8.2 Sequencing
3.9 Bioinformatic Analysis
4 Notes
References
Chapter 9: Nanopore Sequencing to Identify Transposable Element Insertions and Their Epigenetic Modifications
1 Introduction
2 Materials
2.1 Quality Control and Optional Purification and Size Selection of Genomic DNA
2.2 Library Preparation
2.3 Sequencing
2.4 Bioinformatic Analyses
3 Methods
3.1 Quality Control and Optional Purification and Size Selection of Genomic DNA
3.2 ONT Library Preparation
3.3 ONT Sequencing
3.4 From Squiggles to Mapped Reads
3.4.1 Basecalling ONT Data
3.4.2 Genomic Alignment of Reads
3.4.3 DNA Modification Calling
3.5 Downstream Analyses
3.5.1 Polymorphic TE Detection
3.5.2 Visualization of (Non-)reference TEs and Their Methylation Profile
4 Notes
References
Chapter 10: Targeted Nanopore Resequencing and Methylation Analysis of LINE-1 Retrotransposons
1 Introduction
2 Materials
2.1 Guide RNA Synthesis and Purification
2.2 Genomic DNA Extraction
2.3 DNA Library Preparation
2.4 Sequencing
2.5 Data Analysis
3 Methods
3.1 Planning the Sequencing Run
3.1.1 Selection of Target Loci
3.1.2 Design of Guide RNAs
3.2 Prepare Materials Before Library Preparation
3.2.1 sgRNA Synthesis and Purification
3.2.2 Genomic DNA Extraction
3.2.3 Quality Check and Quantification of Genomic DNA
3.3 Library Preparation
3.3.1 Assembly of Cas9 Ribonucleoprotein Particles (RNPs)
3.3.2 Dephosphorylation of the Free 5′ Ends of Genomic DNA
3.3.3 Cas9-Mediated Digestion and dA-Tailing of the Target DNA Region
3.3.4 Quality Check of Cleaved DNA by qPCR Assay
3.3.5 Ligation of Sequencing Adapters
3.3.6 AMPure XP Bead Purification of the DNA Library
3.3.7 Priming of the MinION Flow Cell and Library Loading
3.4 Sequencing
3.5 Data Analysis
3.5.1 Build a Custom Genome
3.5.2 Mapping and Methylation Calling
4 Notes
References
Chapter 11: Inferring Protein-DNA Binding Profiles at Interspersed Repeats Using HiChIP and PAtChER
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Software
3 Methods
3.1 Cell Cross-Linking
3.2 Restriction Digestion
3.2.1 Optional: QC for Restriction Digestion
3.3 Ligation
3.3.1 Optional: QC for Ligation
3.4 Chromatin Immunoprecipitation
3.4.1 Optional: QC for Immunoprecipitation
3.5 Library Generation
3.6 Primary Data Processing
3.6.1 Sequencing Data QC
3.6.2 Mapping
3.7 Data Normalization and Evaluation
4 Notes
References
Chapter 12: Affinity-Based Interactome Analysis of Endogenous LINE-1 Macromolecules
1 Introduction
2 Materials
2.1 Culture and Harvest of N2102Ep Cells
2.2 Cryomilling Cells
2.3 Coupling Antibodies to Magnetic Medium
2.4 Immunoprecipitation for Protein Analyses (Including IP-MS)
2.5 Immunoprecipitation for RNA Analyses (RIP-Seq)
2.6 Protein Mass Spectrometry (MS)
2.7 RNA Sequencing (RIP-Seq)
2.8 Bioinformatics Analyses
2.8.1 Label-Free Quantitative (LFQ) Protein Analysis
2.8.2 Differential Enrichment RNA-seq Analysis
2.8.3 Locus-Specific L1 Quantification
3 Methods
3.1 Cell Culture
3.2 Harvest and Freeze Cells in Liquid Nitrogen
3.3 Cryomilling
3.4 Conjugation of Dynabeads with α-ORF1p Antibody and mIgG
3.4.1 Antibody Buffer Exchange with Microcentrifuge Desalting Columns
3.4.2 Antibody Coupling
3.5 Affinity Capture Using Conjugated Magnetic Medium
3.5.1 Prepare Clarified Cell Extracts
3.5.2 Affinity Capture
3.6 SDS-PAGE and Western Analysis
3.6.1 SDS-PAGE
3.6.2 Western Analysis
3.7 RNA Extraction
3.8 MS Sample Preparation
3.9 Protein Analysis by Mass Spectrometry
3.10 Protein Bioinformatics Analysis
3.10.1 RAW Data Processing Using MQ
3.10.2 Post-processing in R
3.11 RNA Bioinformatics Analysis
3.11.1 RIP-Seq Analysis Steps Are Similar to Typical RNA-Seq Analysis
3.11.2 L1 Locus Detection Using L1EM
4 Notes
Bibliography
Chapter 13: LINE-1 Retrotransposition Assays in Embryonic Stem Cells
1 Introduction
2 Materials
2.1 ESC Lines Used in This Study
2.2 Cell Culture Reagents and Equipment
2.3 Cell Culture Media for Mouse Embryonic Stem Cells (mESCs)
2.4 Cell Culture Media for Human Embryonic Stem Cells (hESCs)
2.5 Reagents and Equipment for Transfecting ESCs
2.6 Plasmids
2.6.1 Plasmids for LINE-1 Cis-Retrotransposition Assay in mESCs
2.6.2 Plasmids for SINE Trans-Tetrotransposition in mESCs
2.6.3 Plasmids for LINE-1 Cis-Retrotransposition Assays in hESCs
2.6.4 Accessory Plasmids Used in hESCs/mESCs to Control Transfection Efficiency and Clonability
2.7 L1 Retrotransposition Reagents and Equipment
3 Methods
3.1 L1NE-1 Cis-Retrotransposition Assays in mESCs
3.1.1 Preparation
3.1.2 Retrotransposition Assay
3.1.3 Quantification of Retrotransposition in Assays Using Selection-Based Cassettes
3.1.4 Quantification of Retrotransposition in Assays Using the EGFP-Based Cassette
3.2 Trans-Retrotransposition Assays in mESCs
3.2.1 Preparation
3.2.2 Procedure
3.3 L1NE-1 Cis-Retrotransposition Assays in hESCs
3.3.1 Preparation
3.3.2 Retrotransposition Assay
3.3.3 Quantification of Retrotransposition in Assays Using Selection-Based Cassettes
3.3.4 Quantification of Retrotransposition in Assays Using the EGFP-Based Cassette
3.3.5 Quantification of Retrotransposition in Assays Using the Fluc-Based Cassette
4 Notes
References
Chapter 14: Experimental Approaches to Study Somatic Transposition in Drosophila Using Whole-Genome DNA Sequencing
1 Introduction
2 Materials
2.1 Drosophila Crosses and Aging
2.2 Tissue Dissection
2.3 DNA Isolation for Short-Read Illumina Sequencing of Individual Tissues
2.4 DNA Isolation for Long-Read ONT Sequencing of Pooled Tissues
2.5 PCR Validation of Candidate TE Insertions
3 Methods
3.1 Standardizing Genetic Backgrounds
3.2 Fly Crossing and Aging
3.3 Tissue Dissection
3.4 Analysis of Individual Tissues Using Short-Read DNA Sequencing
3.4.1 Choice of Genetic Drivers for the Isolation of Clonal Gut Samples
3.4.2 Isolation of Clonal Gut and Head Samples
3.4.3 DNA Isolation for Short-Read Sequencing of Individual Tissues
3.4.4 Low-Input DNA Library Preparation and Short-Read Sequencing
3.4.5 Recommendations for Short-Read Data Analysis (in Galaxy)
3.5 Analysis of Pooled Tissues Using Long-Read DNA Sequencing
3.5.1 Tissue Collection
3.5.2 High-Molecular-Weight (HMW) DNA Isolation for Long-Read Sequencing of Pooled Tissues
3.5.3 Library Preparation and ONT Sequencing
3.5.4 Recommendations for Long-Read ONT Data Analysis
3.6 Validation of Somatic TE Insertions
3.6.1 Manual Validation of Sequencing Reads with IGV
3.6.2 PCR Validation of Candidate Somatic Insertions
4 Notes
References
Chapter 15: Precise and Scarless Insertion of Transposable Elements by Cas9-Mediated Genome Engineering
1 Introduction
2 Materials
3 Methods
3.1 Design and Preparation of sgRNAs by In Vitro Transcription
3.2 Evaluation of sgRNA Efficiency
3.2.1 Cas9 RNP Assembly and Transfection
3.2.2 Editing Efficiency Assessment by TIDE
3.3 Insertion of a Double-Selection Cassette into the Target Locus
3.3.1 Preparation of the Homology-Directed Repair (HDR) Donor Template
3.3.2 Co-transfection of the Cas9 RNP and Donor Plasmid DNA
3.3.3 Initial Detection of Editing Events in Bulk Cell Populations
3.3.4 Single-Cell Cloning
3.3.5 Initial Clone Genotyping
3.3.6 Clone Expansion and Insert Junction Verification
3.3.7 Insert Copy Number Verification
3.3.8 Ganciclovir Sensitivity Test
3.4 Exchange of the HygroTK Cassette by the Transposable Element Sequence
3.4.1 Replacement of the HygroTK Cassette and Selection with Ganciclovir
3.4.2 Single-Cell Cloning to Isolate Clones with a TE Insertion
4 Notes
References
Chapter 16: Epigenetic Manipulation of Transposable and Repetitive Elements
1 Introduction
2 Materials
2.1 TALE Construction
2.2 Dual Luciferase Assay
2.3 TALE Expression and Localization Assays
3 Methods
3.1 TALE Design
3.2 TALE Assembly
3.3 Dual Luciferase Assay to Test TALEs´ Binding to Their DNA Target Sites
3.4 Test TALE Expression and Localization in the Cell Culture System
3.5 Testing TALE Effect on Transcription in Cell Culture System
3.6 Additional Readouts
4 Notes
References
Chapter 17: Genetic Knockout of TE Insertions by CRISPR-Cas9
1 Introduction
2 Materials
2.1 General
2.2 CRISPR of Mammalian Cell Lines
2.3 Screening
3 Methods
3.1 Alt-R CRISPR-Cas9 RNP Complex Formation
3.2 Prepare Cells for Neon Electroporation
3.3 PCR Screening of Bulk CRISPR-Edited Cell Population
3.4 Single-Cell Seeding and Expansion for Alt-R Electroporated Clonal Population Isolation
3.5 Assaying TE KO Clonal Populations for Gene Expression Differences and/or Phenotypes
4 Notes
References
Index