Preface
Contents
Contributors
Chapter 1: Building Phylogenies from Transcriptomic Data
1 Introduction
2 Materials
2.1 Sampling
2.2 Storage
2.3 RNA Extraction
2.4 Library Construction and Sequencing
2.5 Bioinformatic Analysis
3 Methods
3.1 Sampling (See Note 1)
3.2 Storage
3.3 RNA Extraction (See Note 2)
3.4 Library Construction and Sequencing
3.5 Bioinformatic Analysis
3.5.1 Install Conda
3.5.2 Download the Data
3.5.3 Quality Assessment of the Raw Data
3.5.4 Adapter Removing and Filtering (See Note 8)
3.5.5 Assembly with Trinity (See Note 9)
3.5.6 Transcript Clustering
3.5.7 Map Reads Against Assembly
3.5.8 Map Transcripts Against Nucleotide Database
3.5.9 Filter Transcripts with BlobTools
3.5.10 Capture Filtered Transcripts
3.5.11 Translate Transcripts to Protein
3.5.12 Select the Longest Isoform
3.5.13 Orthologue Search
3.5.14 Extract the Protein Sequences of Single-Copy Genes (SC)
3.5.15 Extract the Nucleotide Sequences of Single-Copy Genes (SC)
3.5.16 Alignment (See Note 12)
3.5.17 Trim the Ends
3.5.18 Delete Poorly Aligned Regions (See Note 13)
3.5.19 Concatenation
3.5.20 Phylogenetic Inference
4 Notes
References
Chapter 2: Isolation and Library Preparation of Planarian piRNAs
1 Introduction
2 Materials
2.1 Preparation of Whole-Cell Lysate from Planarian Flatworms
2.1.1 Immunoprecipitation of SMEDWI Proteins and Co-Bound piRNAs
2.1.2 Extraction and Enrichment of piRNA from Planarian Lysate
2.2 Extraction of Co-Immunoprecipitated piRNAs
2.3 Radioactive 5โฒ-End Labeling and Detection of Co-Immunoprecipitated piRNAs
2.4 Adenylation of 3โฒ-Adapter
2.5 piRNA Library Construction
2.5.1 Ligation of Pre-Adenylated 3โฒ-Adapter
2.5.2 Ligation of 5โฒ-RNA Adapter
2.5.3 Reverse Transcription
2.5.4 Small-Scale PCR Amplification
2.5.5 Final PCR Amplification
3 Methods
3.1 Preparation of Whole-Cell Lysate from Planarian Flatworms
3.2 Immunoprecipitation of SMEDWI Proteins and Co-Bound piRNAs
3.3 Extraction of piRNAs from Planarian Lysate
3.3.1 Enrichment of piRNAs from Planarian Lysate by ฮฒ-Elimination
3.3.2 Extraction of Co-Immunoprecipitated piRNAs
3.4 Radioactive 5โฒ-End Labeling and Detection of Co-Immunoprecipitated piRNAs
3.5 Adenylation of the 3โฒ-Adapter
3.6 piRNA Library Construction
3.6.1 Ligation of Pre-Adenylated 3โฒ-Adapter
3.6.2 Ligation of 5โฒ-RNA Adapter
3.6.3 Reverse Transcription
3.6.4 Small-Scale PCR Amplification
3.6.5 Final PCR Amplification
4 Notes
References
Chapter 3: Genome-Wide Analysis of Planarian piRNAs
1 Introduction
2 Materials
3 Methods
3.1 Preprocessing of Sequencing Reads
3.2 Genome Alignment and Annotation of Sequenced piRNAs
3.3 Further Analysis
4 Notes
References
Chapter 4: Combining Fluorescent In Situ Hybridization with Immunofluorescence and Lectin Staining in Planarians
1 Introduction
1.1 Molecular Tools in the Planarian Field
1.2 Method Overview
1.3 Combining Different Molecular Techniques
2 Materials
2.1 Stock Solutions
2.2 FISH Solutions
2.2.1 NAC-FA, Fixation, and Dehydration
2.2.2 Bleaching, Permeabilization, and Hybridization
2.2.3 Post-Hybe Washes and Antibody Incubation
2.2.4 Antibody Washes and TSA Reaction
2.2.5 Double FISH
2.3 Immunofluorescence Solutions
2.3.1 Blocking and Primary Antibody Incubation
3 Methods
3.1 Fluorescent In Situ Hybridization
3.1.1 NAC-FA Fixation and Dehydration
3.1.2 Bleaching, Permeabilization, and Hybridization
3.1.3 Post-Hybe Washes and Antibody Incubation
3.1.4 Antibody Washes and TSA Reaction
3.1.5 Double FISH
3.2 Immunofluorescence Detection/Lectin Staining
3.2.1 Blocking and Primary Antibody Incubation
3.2.2 Primary Antibody Washes, Blocking, and Secondary Antibody Incubation
3.2.3 Secondary Antibody Washes
3.2.4 Clearing and Mounting
4 Notes
References
Chapter 5: Colorimetric Whole-Mount In Situ Hybridization in Planarians
1 Introduction
2 Materials
2.1 Day 1: Mucus Removal, Fixation, and Permeabilization
2.2 Day 2: Bleaching and Incubation with the Riboprobes
2.3 Day 3: Washing and Antibody Incubation
2.4 Day 4: Antibody Washes and Development
3 Methods
3.1 Day 1: Mucus Removal, Fixation, and Permeabilization
3.2 Day 2: Bleaching and Incubation with the Riboprobes
3.3 Day 3: Washing and Antibody Incubation
3.4 Day 4: Antibody Washes and Development
4 Notes
References
Chapter 6: Single-Molecule Fluorescent In Situ Hybridization (smFISH) on Whole-Mount Planarians
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Probe Design and Labelling
2.3 Purification of the Labelled Probes
2.4 Smedwi-1 Immunohistochemistry and smedwi-1/smedwi-2 Double smFISH on Whole-Mount Planarians
3 Methods
3.1 Sample Preparation
3.2 Probe Design and Labelling
3.3 Purification of the Labelled Probes
3.3.1 Probe Validation Using Denaturing PAGE
3.4 Smedwi-1 Immunohistochemistry and smedwi-1/smedwi-2 Double smFISH on Whole-Mount Planarians
4 Notes
References
Chapter 7: Whole-Mount In Situ Hybridization in Large Sexual Schmidtea mediterranea
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 In Situ Hybridization
2.2.1 Colorimetric Development
2.2.2 Fluorescent Development
3 Methods
3.1 Sample Preparation
3.2 In Situ Hybridization
3.2.1 Colorimetric Development
3.2.2 Fluorescent Development
4 Notes
References
Chapter 8: Preparing Planarian Cells for High-Content Fluorescence Microscopy Using RNA in Situ Hybridization and Immunocytoch...
1 Introduction
1.1 Experimental Considerations and Experimental Controls
2 Materials
2.1 Riboprobe Preparation
2.2 Fluorophore-Conjugated Tyramide Synthesis
2.3 Preparing Macerated S. mediterranea
2.4 Fluorescent In Situ Hybridization and Immunocytochemistry
2.4.1 Preparing the Plate(s) and Fixation of Cells
2.4.2 Probe Hybridization and Washing
2.4.3 Riboprobe Development Using Tyramide Signal Amplification
2.4.4 Immunocytochemistry
Staining of Phospho-Histone H3 in Metaphase Nuclei
Staining of Phospho-Histone H3 in Metaphase Nuclei Combined with TSA
Detection of Incorporated BrdU During DNA Synthesis
2.5 Nuclear DNA Staining
3 Methods
3.1 Riboprobe Preparation
3.2 Fluorophore-Conjugated Tyramide Synthesis
3.3 Preparing Macerated Schmidtea mediterranea
3.3.1 Preparing Macerated S. mediterranea for Storage and Later Use
3.3.2 Preparing Macerated S. mediterranea for Subsequent Use
3.4 Fluorescent In Situ Hybridization and Immunocytochemistry
3.4.1 Preparing the Plate(S) and Fixation of Cells
3.4.2 Probe Hybridization and Washing
3.4.3 Riboprobe Development Using Tyramide Signal Amplification
Digoxigenin Riboprobe Development (DIG-Probe)
Fluorescein Riboprobe Development (FL-Probe)
Dinitrophenyl Riboprobe Development (DNP-Probe)
3.4.4 Immunocytochemistry
Staining of Phospho-Histone H3 in Metaphase Nuclei
Staining of Phospho-Histone H3 in Metaphase Nuclei Combined with TSA
Detection of Incorporated BrdU during DNA Synthesis
3.5 Nuclear DNA Staining
4 Notes
Appendix
References
Chapter 9: An RNA/DNA-Based Flow Cytometry Approach for Isolating Slow-Cycling Stem Cells
1 Introduction
2 Materials
2.1 EdU Administration
2.2 Cell Dissociation and Fluorescence-Activated Cell Sorting
2.3 CellMask Staining and Cell Area Quantification
3 Methods
3.1 EdU Administration
3.2 Cell Dissociation and Fluorescence-Activated Cell Sorting
3.3 CellMask Staining and Cell Area Quantification
3.4 EdU Detection
4 Notes
References
Chapter 10: ACME Dissociation-Fixation, Flow Cytometry, and Cell Sorting of Freshwater Planarian Cells
1 Introduction
2 Materials
2.1 ACME Dissociation-Fixation
2.2 ACME Cell Imaging and Sorting
3 Methods
3.1 ACME Dissociation-Fixation
3.2 ACME Cell Imaging and Sorting
4 Notes
References
Chapter 11: Papain-Based Dissociation of Schmidtea mediterranea Cells
1 Introduction
2 Materials
2.1 Mucus Removal and Digestion Pretreatment
2.2 Cell Dissociation
3 Methods
3.1 Mucus Removal and Digestion Pretreatment
3.2 Cell Dissociation
4 Notes
References
12: Live Immunostaining and Flow Cytometry of Schmidtea Mediterranea Cells
1 Introduction
2 Materials
2.1 Live Immunostaining
2.2 Flow Cytometry and Cell Sorting
3 Methods
3.1 Live Immunostaining
3.1.1 Single Live Immunostaining
3.1.2 Double Live Immunostaining (Non-Cross-Reacting Secondary Antibodies)
3.1.3 Double Live Immunostaining (Cross-Reacting Secondary Antibodies)
3.2 Flow Cytometry and Cell Sorting
4 Notes
References
Chapter 13: Live Imaging in Planarians: Immobilization and Real-Time Visualization of Reactive Oxygen Species
1 Introduction
2 Materials
2.1 Detection of Reactive Oxygen Species
2.2 Immobilization
2.3 Live Imaging
2.4 Recovery of the planarian
3 Methods
3.1 Detection of Reactive Oxygen Species
3.2 Immobilization
3.3 Live Imaging
3.4 Recovery of the Planarian
4 Notes
References
Chapter 14: A Planarian Model System to Study Host-Pathogen Interactions
1 Introduction
2 Materials
2.1 Culturing of C. Albicans for Planarian Infection
2.2 Infecting Planarians Through Injection
2.3 Infecting Planarians Through Feeding
2.4 Infecting Planarians Through Soaking
2.5 Fixation After Infection Via Feeding
2.6 Fixation After Infection Via Injection and Soaking
2.7 Whole-Mount Immunohistochemistry
2.8 Calculating C. albicans Concentration from Overnight Cultures
3 Methods
3.1 Culturing C. albicans for Planarian Infections
3.2 Infecting Planarians Through Injection
3.3 Infecting Planarians Through Feeding
3.4 Infecting Planarians Through Soaking
3.5 Fixation After Infection Via Feeding
3.6 Fixation After Infection Via Soaking or Injection
3.7 Whole-Mount Immunohistochemistry
3.8 Calculating C. albicans Concentration from Overnight Cultures
4 Notes
References
Chapter 15: TUNEL Staining in Sections of Paraffin-Enabled Planarians
1 Introduction
2 Materials
2.1 Planarian Fixation
2.2 Paraffin Embedding, Sectioning, and Deparaffinization
2.3 Tunnel Staining Using ApopTag Red in Situ Apoptosis Detection Kit
3 Methods
3.1 Planarian Fixation
3.2 Paraffin Embedding, Sectioning, and Deparaffinization
3.3 Tunnel Staining Using ApopTag Red in Situ Apoptosis Detection Kit
4 Notes
References
Chapter 16: Quantitative Analysis of Planarian Pigmentation
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 17: mRNA Transfection of S. mediterranea for Luminescence Analysis
1 Introduction
2 Materials
2.1 In Vitro Transcription
2.2 Dissociation of Planaria into Individualized Cells
2.3 In Vitro Cell mRNA Transfection
2.4 Dissociated Cell Luminescence Analysis
2.5 Live Worm mRNA Transfection by Injection
2.6 Luminescence Analysis of Lysed Transfected Worms by a Plate Reader
2.7 Luminescence In Vivo Imaging of mRNA Transfected Worms
3 Methods
3.1 In Vitro Transcription
3.2 Dissociation of Planaria into Individualized Cells
3.3 In Vitro Cell mRNA Transfection
3.4 Dissociated Cell Luminescence Analysis
3.5 Live Worm mRNA Transfection by Injection
3.6 Luminescence Analysis of Lysed Transfected Worms by a Plate Reader
3.7 Luminescence In Vivo Imaging of mRNA Transfected Worms
4 Notes
References
Index