Preface
Contents
Contributors
Part I: Experimental Protocols
Chapter 1: Monitoring Bacterial Translation Rates Genome-Wide
1 Introduction
2 Materials
2.1 Cell Harvesting by Rapid Filtration
2.2 Cell Pulverization
2.3 mRNA Enrichment and Fragmentation
2.4 Extract Preparation, MNase Footprinting, and Monosome Isolation
2.5 RNA Extraction
2.6 Footprint Size Selection
2.7 Dephosphorylation
2.8 Linker Ligation
2.9 Reverse Transcription
2.10 Circularization
2.11 rRNA Subtraction
2.12 Library Amplification by PCR
2.13 Quantify, Sequence, and Analyze
3 Methods
3.1 Cell Harvesting by Rapid Filtration (~10-15 min for Harvesting)
3.2 Cell Pulverization (~1 h)
3.3 mRNA Enrichment (~4-5 h) and Fragmentation (~1.5 h)
3.4 Extract Preparation (~30 min), MNase Footprinting (~1.5 h), and Monosome Isolation (~3.5 h)
3.5 RNA Extraction (~2 h)
3.6 Footprint Size Selection (~1.5 h)
3.7 Dephosphorylation (~2.5 h)
3.8 Linker Ligation (~3 h)
3.9 Reverse Transcription (~3.5 h)
3.10 Circularization (~2.5 h)
3.11 rRNA Subtraction (~3 h)
3.12 Library Amplification by PCR (~2.5 h)
3.13 Quantify, Sequence, and Alignment
4 Notes
References
Chapter 2: Identification of Translation Start Sites in Bacterial Genomes
1 Introduction
2 Materials
2.1 Metabolic Labeling with RET
2.2 Preparation of Cell Lysates for Ribo-RET
2.3 Pulverization of Cells
2.4 Preparation of Cell Lysate an of Ribosome-Protected mRNA Fragments
2.5 Monosome Isolation by Sucrose Gradient Centrifugation
2.6 Phenol-Chloroform Extraction of Ribosome-Protected mRNA Fragments
2.7 Size Selection of Ribosome-Protected mRNA Fragments
2.8 Dephosphorylation
2.9 Enzymatic Pre-adenylation of Linkers and Linker Ligation
2.10 Reverse Transcription
2.11 Circularization
2.12 PCR Amplification
3 Methods
3.1 Optimization of RET Treatment
3.2 Collection of Cells Treated with RET
3.3 Preparation of Cell Lysates
3.4 Preparation of Ribosome-Protected mRNA Fragments
3.5 Isolation of Monosomes by Sucrose Gradient Fractionation
3.6 Phenol-Chloroform Extraction of Ribosome-Protected mRNA Fragments
3.7 Size Selection of Ribosome-Protected mRNA Fragments
3.8 Dephosphorylation of Ribosome-Protected mRNA Fragments
3.9 Enzymatic Adenylation of the Linkers and Linker Ligation
3.10 Reverse Transcription
3.11 Circularization of cDNA
3.12 PCR Amplification
3.13 Preparing Samples for Next Generation Sequencing
3.14 Computational Processing of Ribosome Profiling Reads
4 Notes
References
Chapter 3: Genome-Wide Ribosome Profiling of the Plasmodium falciparum Intraerythrocytic Developmental Cycle
1 Introduction
2 Materials
2.1 Preparation of Parasite Lysates
2.2 Micrococcal Nuclease Digestion
2.3 Sucrose Gradients
2.4 RNA Isolation from Monosome Sucrose Gradient Fractions
2.5 Total RNA Isolation from iRBCs
2.6 polyA+ mRNA Purification
2.7 polyA+ mRNA Zn-mediated Fragmentation
2.8 Fragmented mRNA and Ribosome Footprint Size Selection
2.9 Rapid Gel Extraction
2.10 3β² End Dephosphorylation
2.11 Linker Ligation
2.12 Size Selection of the Linker-ligated Product
2.13 Ribosomal RNA Subtraction
2.14 Reverse Transcription
2.15 Circularization
2.16 PCR Amplification
3 Methods
3.1 Preparation of Parasite Lysates
3.2 Micrococcal Nuclease Digestion
3.3 Sucrose Gradients
3.4 RNA Isolation from Monosome Sucrose Gradient Fractions
3.5 Total RNA Isolation from iRBCs
3.6 PolyA+ mRNA Purification
3.7 PolyA+ mRNA Zn-mediated Fragmentation
3.8 Fragmented mRNA and Ribosome Footprint Size Selection
3.9 Rapid Gel Extraction
3.10 3β² End Dephosphorylation
3.11 Linker Ligation
3.12 Size Selection of the Linker-ligated Product
3.13 Ribosomal RNA Subtraction
3.14 Reverse Transcription
3.15 Circularization
3.16 PCR Amplification
3.17 Sequencing and Data Analysis
4 Notes
References
Chapter 4: Performing Ribosome Profiling to Assess Translation in Vegetative and Meiotic Yeast Cells
1 Introduction
2 Materials
2.1 Media
2.2 Base Reagents and Materials
2.3 Non-standard Equipment or Facilities Required
2.4 Buffers and Solutions
2.5 Oligonucleotides
3 Methods
3.1 Yeast Growth and Sporulation Conditions (See Note 18)
3.2 Cell Harvesting and Preparing Polysome Lysis Buffer (See Notes 19 and 20)
3.3 Yeast Cell Lysis (See Notes 11 and 21)
3.4 Extract Preparation (See Note 22)
3.5 Footprint Isolation by Sucrose Gradient (See Note 23)
3.6 RNA Extraction from Monosome Fraction (See Note 24)
3.7 FP Size Selection
3.8 Gel Staining and Imaging (See Notes 16 and 25)
3.9 Gel Extraction (See Note 26)
3.10 rRNA Subtraction (See Notes 9, 14, and 28)
3.11 Dephosphorylation and Linker Ligation (See Note 27)
3.12 Reverse Transcription (See Note 14)
3.13 Circularization (See Note 14)
3.14 PCR Amplification (See Notes 14, 29 and 30)
3.15 TapeStation Analysis for Quality Control Assessment (See Note 10)
4 Notes
References
Chapter 5: Quantitative Comparisons of Translation Activity by Ribosome Profiling with Internal Standards
1 Introduction
2 Materials
2.1 Preparation of HeLa Lysate
2.2 Preparation of Saccharomyces cerevisiae Lysate
2.3 Quantification of Ribosomal RNA
2.4 RNase Digestion, Addition of Internal Standard, and Footprint Isolation from Sucrose Gradients
2.5 Small RNA Enrichment, Dephosphorylation, and Footprint Size Selection
2.6 3β² Adapter Ligation and Ribosomal RNA Depletion
2.7 cDNA Synthesis and Circularization
2.8 PCR and Preparation for Sequencing
3 Methods
3.1 Preparation of HeLa Lysate
3.2 Preparation of Saccharomyces cerevisiae Lysate
3.3 Quantification of Ribosomal RNA
3.4 RNase Digestion, Addition of Internal Standard, and Footprint Isolation from Sucrose Gradients
3.5 Small RNA Enrichment, Dephosphorylation, and Footprint Size Selection
3.6 3β² Adapter Ligation and Ribosomal RNA Depletion
3.7 cDNA Synthesis and Circularization
3.8 PCR and Preparation for Sequencing
3.9 Data Analysis
4 Notes
References
Chapter 6: Genome-Wide Analysis of Translation in Replicatively Aged Yeast
1 Introduction
2 Materials
2.1 Preparation of Worm Lysate
2.2 Isolation of Replicatively Aged Yeast Cells
2.3 Counting Bud Scars
2.4 Preparation of Yeast Lysate
2.5 Footprint Extraction
2.6 Footprint Fragment Purification
2.7 Poly(A) mRNA Extraction
2.8 Dephosphorylation
2.9 3β²-Adapter Ligation
2.10 Reverse Transcription
2.11 Circularization and PCR Library Amplification
3 Methods
3.1 Preparation of Worm Lysate
3.2 Isolation of Replicatively Aged Yeast Cells
3.3 Counting Bud Scars
3.4 Preparation of Yeast Lysate
3.5 Footprint Extraction
3.6 Footprint Fragment Purification
3.7 Poly(A) mRNA Extraction and mRNA Fragmentation
3.8 Dephosphorylation
3.9 3β²-Adapter Ligation
3.10 Reverse Transcription
3.11 Circularization
3.12 PCR Library Amplification
3.13 Library Quantification and High-Throughput Sequencing
4 Notes
References
Chapter 7: Tissue-Specific Ribosome Profiling in Drosophila
1 Introduction
2 Materials
3 Methods
3.1 Sample Collection
3.2 Generation of Ribosome Protected mRNA Fragments (Ribosome Footprints)
3.3 Phosphatase Treatment and 3β² Ligation for Library Generation
3.4 Kinase Treatment, 5β² Ligation and Reverse Transcription for Library Generation
3.5 Library PCR Amplification and Purification
3.6 Recommendations for Next-Generation Sequencing and Data Analysis
4 Notes
References
Chapter 8: Measuring Organ-Specific Translation Elongation Rate in Mice
1 Introduction
2 Materials
2.1 Animal Injections
2.2 Tissue Lysis
2.3 Sucrose Gradients Fractionation
2.4 Footprint Extraction and Library Preparation
2.5 Adapter and PCR Primer Sequences
3 Methods
3.1 Animal Injections
3.2 Tissue Lysis
3.3 Sucrose Gradient Fractionation
3.4 Footprint Extraction and Library Preparation
3.5 Data Analysis
4 Notes
References
Chapter 9: Active Ribosome Profiling with RiboLace: From Bench to Data Analysis
1 Introduction
2 Materials
2.1 Preparation of Cell Lysates
2.2 Bead Functionalization
2.3 Endonuclease Digestion
2.4 Isolation of Active Ribosomes and Purification of Ribosome-Protected Fragments (RPFs)
2.5 RPF PAGE Purification
2.6 T4 Polynucleotide Kinase (PNK) Dephosphorylation
2.7 Adaptor Ligation and PAGE Purification
2.8 Reverse Transcription and cDNA PAGE Purification
2.9 cDNA Circularization
2.10 PCR Amplification and PAGE Purification of the Library
3 Methods
3.1 Preparation of Cell Lysates for Active-ribo-seq (Estimated Time, 25 min)
3.2 Bead Functionalization (Estimated Time, 90 min)
3.3 Endonuclease Digestion (Estimated Time, 90 min)
3.4 Isolation of Active Ribosomes and Purification of RPFs (Estimated Time, 3 h. Overnight Optional)
3.5 RPF PAGE Purification
3.6 T4 Polynucleotide Kinase Dephosphorylation
3.7 Adaptor Ligation and PAGE Purification
3.8 Reverse Transcription and cDNA PAGE Purification
3.9 cDNA Circularization
3.10 PCR Library Amplification and Library PAGE Purification
3.11 RPF Sequencing and Data Processing
3.12 Positional Analysis with riboWaltz
4 Notes
References
Chapter 10: Poly-A Tailing and Adaptor Ligation Methods for Ribo-Seq Library Construction
1 Introduction
2 Materials
2.1 Buffers
2.2 Reagents
2.3 Primers and Linkers for Ribo-seq Library Construction
2.4 Equipment
3 Methods
3.1 Cell Lysis for Ribosome Profiling
3.2 Polysome Gradient
3.3 RNase I Digestion of Ribosome Fractions
3.4 cDNA Library Construction (Poly-A Tailing Method)
3.4.1 RNA Dephosphorylation
3.4.2 RNA Size Selection and Extraction
3.4.3 Ethanol Precipitation
3.4.4 Poly-A Tailing
3.4.5 cDNA Synthesis
3.4.6 Size Selection and DNA Extraction
3.4.7 Circularization
3.4.8 Linearization
3.4.9 Size Selection and DNA Extraction
3.4.10 PCR Amplification and Size Selection
3.5 cDNA Library Construction (Adaptor Ligation Method)
3.5.1 RNA Size Selection and Extraction
3.5.2 RNA Dephosphorylation
3.5.3 Linker Ligation
3.5.4 RNA Size Selection and Extraction
3.5.5 cDNA Synthesis
3.5.6 Size Selection and DNA Extraction
3.5.7 Circularization
3.5.8 PCR Amplification and Size Selection
3.6 Deep Sequencing
3.7 Sequencing Data Analysis
4 Notes
References
Chapter 11: Choice of Ribonucleases for Ribosome Profiling Experiments
1 Introduction
1.1 Ribonucleases and Their Properties
2 Materials
2.1 Ribonucleases
2.2 Other Reagents
2.3 Sucrose Gradients
2.4 Subtractive Hybridization
3 Methods
3.1 Test Ribosome Stability in the Presence of Ribonucleases
3.2 Quantify the Number of Monosomes and Polysomes
3.3 Fine-Tuning Nuclease Digestion Conditions
3.4 Improve Footprints Yield by Subtractive Hybridization
4 Notes
References
Part II: Bioinformatics and Statistical Analysis
Chapter 12: Codon Resolution Analysis of Ribosome Profiling Data
1 Introduction
2 Materials
2.1 Software Dependencies
2.2 Download and Prepare the Pipeline
2.3 Genome and Sequencing Data
2.4 Example Sequencing Data and Code
2.5 Hardware Requirements
3 Methods
3.1 [Optional] Sequencing Read Pre-processing and Mapping
3.2 Statistics and Filtering Ribosomal Reads by Length
3.3 RPF Coverage Within Start and Stop Codon Regions
3.4 Calibration of RPFs
4 Notes
References
Chapter 13: Assessing Ribosome Distribution Along Transcripts with Polarity Scores and Regression Slope Estimates
Abbreviations
1 Introduction
2 Materials
2.1 Overview of Test Data
2.2 Software Requirements
2.2.1 Setting Up Necessary Tools and Packages
2.2.2 Setting Up Conda Environment
2.3 Preparing Test Data
2.4 Ribo-Seq Read Processing
2.4.1 Preparatory Step
2.4.2 Trimming and Alignment
3 Methods
3.1 Basic Preprocessing
3.1.1 Preparing Transcript Annotation
3.1.2 Preparing Coverage Profiles
3.2 Polarity Score
3.2.1 Estimating the Scores
3.2.2 Transcript List Filtering
3.2.3 Finalizing Polarity Score Lists
3.2.4 Polarity Z-Score Estimation
3.2.5 Visualizing Per-Sample Score Distribution
3.3 Segmentation and Relative Slope Estimation
3.3.1 Simplifying Coverage Profiles
3.3.2 Clipping Segmentation to Coding Segments
3.3.3 Generation of Flattened Coverage Profiles
3.3.4 Estimating Segmentation-Based Comparative Metrics
3.3.5 Filtering Results
3.3.6 Estimating Z-Scores
3.3.7 Visualizing Per-Sample Distributions
4 Notes
4.1 Notes on Software Usage
4.2 Notes on Ribo-Seq Data Analysis
References
Chapter 14: Unsupervised Bayesian Prediction of RNA Translation from Ribosome Profiling Data
1 Introduction
2 Materials
2.1 Hardware
2.2 Software
2.3 Data Sources
3 Methods
3.1 Quality Control
3.2 Creating Reference Genome Indices
3.2.1 Output Files
3.2.2 A Note on ORF Labels
3.3 Running the Rp-Bp Pipeline
3.4 Creating the ORF Profiles
3.4.1 Output Files
3.5 Predicting Translated ORFs
3.5.1 Output Files
3.6 Analysis
3.6.1 Quality Control and Periodicity Estimation
3.6.2 Output Files
3.6.3 ORF Predictions
4 Notes
References
Chapter 15: uORF-seqr: A Machine Learning-Based Approach to the Identification of Upstream Open Reading Frames in Yeast
1 Introduction
1.1 Overview of Workflow
1.2 P-site Fractionation for Reading Frame Determination
1.3 Regression Feature Definition
1.4 uORF-seqr Regression and Statistical Control
2 Materials
2.1 Data
2.2 Computational Resources
2.3 R Requirements
2.4 Python Package Requirements
3 Methods
3.1 Installation of uORF-seqr
3.2 Preprocess a New Reference Genome
3.3 Quantify Features for a New Experiment
3.3.1 Short Descriptions of Arguments
3.4 Predict Candidate uORFs Based on Calculated Features
3.5 Interpreting Results of uORF Prediction
4 Notes
References
Chapter 16: Genome-Wide Analysis of Actively Translated Open Reading Frames Using RiboTaper/ORFquant
1 Introduction
Box 1:
2 Materials
3 Methods
3.1 Preparing Annotation Files for RiboTaper
3.2 Preparing Annotation Files for ORFquant
3.3 Preparing P-Site Signal Data
3.4 Running RiboTaper
3.5 Running ORFquant
3.6 Plotting, Interpreting, and Filtering Results
3.7 Caveats to Interpretation
4 Notes
References
Index