Preface
Contents
Contributors
Part I: Transcriptomic Analyses
Chapter 1: Single-Cell RNA Sequencing in Yeast Using the 10x Genomics Chromium Device
1 Introduction
2 Materials
2.1 Growth Media and Sampling Buffers
2.2 In-Droplet Spheroplasting and Cell Lysis
2.3 Software Installations
3 Methods
3.1 Sample Preparation
3.2 Single-Cell Library Preparation
3.3 Cell Ranger Pipeline
3.3.1 Demultiplex Raw Base Call (BCL) into FASTQ Files
3.3.2 Mapping to Reference Genome and UMI Counting
3.3.3 Aggregate Data from Multiple Samples
3.4 Secondary Analysis in R
3.4.1 Read-In of the Data, QC and Filtering, Normalization
3.4.2 Dimensional Reduction and Visual Exploration of Data
3.4.3 Clustering and Differential Gene Expression
4 Notes
References
Chapter 2: Genome-Wide Profiling of Transcription Initiation with STRIPE-seq
1 Introduction
2 Materials
2.1 Reagents and Buffers
2.2 Oligonucleotides
2.3 Equipment
3 Methods
3.1 Depletion of Uncapped RNA
3.2 Template-Switching Reverse Transcription
3.3 Library PCR
3.4 Small Fragment Removal
3.5 Large Fragment Removal
3.6 Computational Analysis
4 Notes
References
Chapter 3: A Modified Cross-Linking Analysis of cDNAs (CRAC) Protocol for Detecting RNA-Protein Interactions and Transcription...
1 Introduction
2 Materials
2.1 Yeast Strain and Media
2.2 Reagents and Enzymes
2.3 Buffers
2.4 Equipment
2.5 Oligonucleotides
2.5.1 3β² Linkers
2.5.2 5β² Linker
2.5.3 RT and PCR Oligonucleotides
2.6 Software and Algorithms
3 Methods
3.1 Yeast Growth and UV Cross-Linking
3.2 Preparation of Cell Lysate
3.3 Affinity Purification of the Complex Using IgG Beads
3.4 Partial RNase Digestion
3.5 Affinity Purification of the Complex Using Nickel Beads and on-Beads Linker Ligation
3.5.1 Binding of Protein-RNA Complexes
3.5.2 On-Beads 3β²-End Dephosphorylation (Optional)
3.5.3 On-Beads 3β² Linker Ligation
3.5.4 On-Beads 5β²-End Phosphorylation
3.5.5 On-Beads 5β² Linker Ligation
3.5.6 Purification of Protein-RNA Complexes
3.6 RNA Isolation
3.7 Synthesis of the cDNA Library
3.8 Amplification of the cDNA Library
3.8.1 Estimate of the Minimal Number of Cycles for Amplifying the cDNA Library
3.8.2 Analysis of the Size Profile of cDNA Library
3.9 Final PCR and Library Purification
3.10 Data Analysis
4 Notes
References
Chapter 4: High-Resolution Deep Sequencing of Nascent Transcription in Yeast with BioGRO-seq
1 Introduction
2 Materials
2.1 Equipment
2.2 Nascent RNA Biotinylation by Run-on
3 Methods
3.1 Preparation of DNase-Free RNase A
3.2 Preparing Dynabeads for RNA Binding
3.3 BioGRO-seq Method
3.4 RNA Extraction
3.5 Streptavidin Paramagnetic Bead Pull-Down of Biotinylated RNA
3.6 End Repair and Library Prep for Deep Sequencing
3.7 Total RNA Extraction for Conventional Single-Gene or Transcriptomic Analysis (Optional, See Note 17)
4 Notes
References
Chapter 5: Direct Sequencing of RNA and RNA Modification Identification Using Nanopore
1 Introduction
2 Materials
2.1 Oxford Nanopore TechnologiesΒ΄ Kits
2.2 Enzymes, Buffers, Water
2.3 Other Reagents and Consumables
2.4 Equipment
3 Methods
3.1 Poly(A) Tailing Reaction
3.2 dRNA-seq
4 Notes
References
Chapter 6: UNAGI: Yeast Transcriptome Reconstruction and Gene Discovery Using Nanopore Sequencing
1 Introduction
2 Materials
2.1 Generating Full-Length cDNA and Library Preparation
2.2 Software
2.3 System Requirement
3 Methods
3.1 Generating Full-Length cDNA
3.2 Library Preparation
3.2.1 End-Prep
3.2.2 Barcoding
3.2.3 Pooling and Adapter Ligation
3.2.4 Priming and Loading on the Flow Cell
3.3 UNAGI Pipeline
3.3.1 Setting Up Software
3.3.2 Trimming Adapter
3.3.3 Running UNAGI
4 Notes
References
Chapter 7: Identification of Taxonomically Restricted Transcripts from Illumina RNA Sequencing Data
1 Introduction
2 Materials
2.1 Growth Media
2.2 Culture Preparation
2.3 RNA Purification
2.4 Library Preparation
2.5 Bioinformatic Pipeline
3 Methods
3.1 Culture Preparation
3.2 RNA Purification
3.3 Library Preparation
3.4 Sequencing
3.5 Bioinformatic Pipeline (Fig. 1)
3.5.1 Quality Control of Raw Sequencing Data
3.5.2 Construct Robust Annotations
3.5.3 Determine Taxonomic Conservation of Transcripts
4 Notes
References
Part II: DNA Replication and Protein/DNA Interactions
Chapter 8: FORK-seq: Single-Molecule Profiling of DNA Replication
1 Introduction
2 Materials
2.1 Yeast Strain
2.2 Yeast Culture and Labelling
2.3 DNA Extraction
2.4 Sequencing Libraries
2.5 Sequencing
3 Methods
3.1 Yeast Culture
3.2 Replication Labelling
3.3 DNA Extraction
3.3.1 Cell Wall Lysis
3.3.2 Spheroplasts Lysis and Protein Precipitation
3.3.3 DNA Precipitation
3.3.4 Elution, Quantification and Quality Control
3.4 Nanopore Sequencing Libraries
3.4.1 DNA Repair and Purification
3.4.2 Native Barcode Ligation-Facultative
3.4.3 Adapter Ligation
3.4.4 Priming the Flow Cell and Loading the Library
3.5 Nanopore Sequencing and Base Calling
3.5.1 Concomitant Acquisition and Base Calling
3.5.2 Acquisition with No Base Calling
3.5.3 Base Calling with Guppy Independently of Data Acquisition
3.5.4 Demultiplexing of the FAST5 Files
3.5.5 Concatenate FASTQ Files
3.6 Data Analysis with RepNano
3.6.1 Installation
3.6.2 BrdU Detection
3.6.3 Fork Detection
4 Notes
References
Chapter 9: CUT&RUN Profiling of the Budding Yeast Epigenome
1 Introduction
2 Materials
2.1 Yeast Culture and Nuclei Preparation
2.2 CUT&RUN and Sequencing
2.3 General Equipment
3 Method
3.1 Yeast Nuclei Preparation
3.2 CUT&RUN
3.3 Native ChIP with CUT&RUN Supernatant as Input (CUT&RUN.ChIP)
3.4 Library Preparation and Paired-End Illumina Sequencing
3.5 Data Processing and Analysis
4 Notes
References
Chapter 10: ChIP-SICAP: A New Tool to Explore Gene-Regulatory Networks in Candida albicans and Other Yeasts
1 Introduction
2 Materials
2.1 Strains and Growth Conditions
2.2 Crosslinking and Cell Lysis
2.3 Chromatin Fragmentation and Immunoprecipitation
2.4 Biotinylation and Capture of Coenriched Chromatin
2.5 Stringent Washing Protocol
2.6 Protein Digestion and Sample Preparation for MS
2.7 Chromatin Recovery and Preparation for NGS
2.8 Data Processing and Analysis
2.9 Software
3 Methods
3.1 Culturing and Cross-Linking
3.2 Dynabead-Antibody Preparation
3.3 Cell Lysis, Chromatin Shearing and Immunoprecipitation
3.4 Biotinylation of Enriched Chromatin
3.5 Removal of Protein Contaminations
3.6 On Bead Digestion of Copurified Proteins
3.7 Sample Preparation and LC-MS/MS Acquisition
3.8 Qualitative LC-MS/MS Data Analyses
3.9 Recovery of Chromatin Fragments and Preparation for NGS
3.10 NGS Data Analyses
3.10.1 Quality Control Assessment and Adapter Trimming
3.10.2 Mapping of the Reads to the Reference Genome
3.10.3 Peak-Detection
3.10.4 Motif Discovery
4 Notes
References
Part III: Translation Dynamics, Protein Complexes, and Proteomics
Chapter 11: Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling
1 Introduction
2 Materials
3 Methods
3.1 Cell Growth, Collection, and Lysis
3.2 Total Translatome Extraction and Affinity Purification of GFP Tagged Subunits (See Note 2)
3.3 Preparation of a Deep Sequencing Library, in RNA Safe Conditions (See Note 4)
3.3.1 Acid Phenol Extraction of RNA
3.3.2 Quantify Total RNA Concentration by Nanodrop
3.3.3 Gel-Purify Ribosome Protected Footprint Fragments
3.3.4 Dephosphorylate 3β² Ends of Gel-Purified Fragments
3.3.5 Quantify Concentration by Bioanalyzer/TapeStation
3.3.6 Ligate 3β² End with Linker-1
3.3.7 Gel-Purify 3β² Linked Footprint Fragments
3.3.8 Recovering 3β² Linked Footprint RNA Fragments from Gel
3.3.9 Reverse Transcribe 3β² Linked Footprint Fragments to Generate ssDNA
3.3.10 Recovering DNA Fragments from Gel
3.3.11 Circularize ssDNA
3.3.12 PCR Amplify
3.3.13 Analysis and Quantification of the cDNA Library Products
3.3.14 Next-Generation Sequencing by HiSeq\NextSeq\NovaSeq
3.4 Data Analysis
4 Notes
References
Chapter 12: Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevis...
1 Introduction
2 Materials
2.1 Conjugation of Magnetic Dynabeads with Rabbit IgG (See Note 1)
2.2 Harvesting and Freezing Yeast Cells
2.3 Cryolysis (Cryomilling) (See Note 4)
2.4 Weighing Yeast Cell Grindate
2.5 Single-Step Affinity Purification
2.6 Trypsin Digest
2.7 Mass Spectrometry
2.8 Sample Analysis
3 Methods
3.1 Conjugation of Magnetic Dynabeads with Rabbit IgG
3.2 Harvesting and Freezing of Cells
3.3 Cryogenic Cell Lysis (Cryo-Milling)
3.4 Single-Step Affinity Purification (See Note 18)
3.4.1 Weighing the Yeast Cell Grindate
3.4.2 Affinity Purification
3.5 On-Beads Trypsin Digestion
3.5.1 Preparations
3.5.2 On-Bead Digestion
3.6 Protein Elution for SDS-PAGE Staining and Western Blotting
3.7 Mass Spectrometry
3.7.1 C18 ZipTip Sample Cleanup
3.7.2 Resolubilizing the Dried Peptides for LC-MS Injection
3.7.3 Sample QC and Sample Quantity Determination for LC-MS Injections
3.7.4 LC-MS Method and Sample Injection
3.8 Data Processing and Analysis
3.8.1 Data Processing
3.8.2 Data Analysis
4 Notes
References
Chapter 13: Composition and Dynamics of Protein Complexes Measured by Quantitative Mass Spectrometry of Affinity-Purified Samp...
1 Introduction
2 Materials
2.1 Yeast Growth Medium
2.2 Magnetic Beads Preparation
2.3 Cell Lysis and Purification
2.4 Chromatography and Mass Spectrometry
3 Methods
3.1 Preparation of IgG Coupled Superparamagnetic Beads
3.2 Preparation of a Total Yeast Extract
3.3 Purification of Tagged Proteins
3.4 Protein Precipitation with Methanol-Chloroform (See Note 4)
3.5 Proteolytic Lysis of Proteins for Mass-Spectrometry
3.5.1 Reduction and Alkylation
3.5.2 Protein Digestion
3.5.3 Peptides Desalting
3.6 Liquid Chromatography Coupled with Mass Spectrometry
3.7 Identification of Proteins and Raw Quantitation of Results
3.8 Interpretation of Quantitative MS Results
4 Notes
References
Chapter 14: Deep Mutational Scanning of Protein-Protein Interactions Between Partners Expressed from Their Endogenous Loci In ...
1 Introduction
2 Materials
2.1 Strain Preparation (Stuffing/Gene Deletion/Gene Tagging) (See Note 1)
2.2 Library Preparation
2.3 Yeast CRISPR-Cas9 Genomic Integration
2.4 Liquid PCA
3 Methods
3.1 Strain Preparation
3.2 Library Preparation
3.3 Yeast Integration
3.4 Liquid Dihydrofolate Reductase Protein-Fragment Complementation Assay (DHFR-PCA) (See Fig. 3)
4 Notes
References
Chapter 15: Proteomic Mapping by APEX2-Catalyzed Proximity Labeling in Saccharomyces cerevisiae Semipermeabilized Cells
1 Introduction
2 Materials and Equipment
2.1 Yeast Media
2.2 Transformed Yeast Cells, Carrying APEX2 Constructs
2.3 Storage of Semipermeabilized Yeast Cells for Proximity Labeling
2.4 Proximity Labeling, Detection by Western Blotting, Affinity Purification for Mass Spectrometry Analysis
3 Methods
3.1 Preparing Semipermeabilized Yeast Samples for PL and Western Blot Analysis
3.2 Small Scale Proximity Labeling for Western Blotting
3.3 Preparing Semipermeabilized SILAC Samples for PL and Mass Spectrometry Proteomics
3.4 Three-State Proximity Labeling for MS Proteomics
3.5 MS Proteomics and MS Data Analysis
4 Notes
References
Chapter 16: Quantitative Proteomics in Yeast: From bSLIM and Proteome Discoverer Outputs to Graphical Assessment of the Signif...
1 Introduction
2 Materials
3 Methods
3.1 Cell Growth and Preparation of Protein Extracts
3.2 Data Processing Workflows
3.2.1 Proteome Discoverer Analysis
3.2.2 Isotopolog Intensity Extraction and Peptide/Protein Quantification Using a Dedicated bSLIM KNIME Workflow (Fig. 4)
3.2.3 Statistics and Graphical Assessment of Score Significance
3.3 Case Study: BY4742 Vs S288c Proteome Comparison
4 Notes
References
Chapter 17: A Strong Cation Exchange Chromatography Protocol for Examining N-Terminal Proteoforms
1 Introduction
2 Materials
2.1 pGAPase Purification
2.2 Cell Lysis and Protein Extraction
2.3 Sample Preparation for N-Terminal Peptide Enrichment
2.4 Pyroglutamate Removal
2.5 Strong Cation Exchange (SCX) Enrichment of N-Terminal Peptides
2.6 Oxidation and Cleanup of Purified N-Terminal Peptides
3 Methods
3.1 pGAPase Purification
3.2 Cell Lysis and Protein Extraction
3.2.1 Yeast Cell Wall Disruption
3.2.2 Sample Preparation Using Guanidinium Hydrochloride
3.2.3 Sample Preparation Using APols
3.3 Sample Preparation for N-Terminal Peptides Enrichment
3.4 Pyroglutamate Removal
3.5 Strong Cation Exchange (SCX) Enrichment of N-Terminal Peptides
3.6 Oxidation and Clean-up of Purified N-Terminal Peptides
3.7 Data Analysis
3.7.1 Search Settings for N-Terminally Acetylated Peptides
3.7.2 Search Settings for Peptides Other Than N-Terminally Acetylated Peptides
4 Notes
References
Part IV: Genotypic Screens and Phenotypic Profiling
Chapter 18: RNA Interference (RNAi) as a Tool for High-Resolution Phenotypic Screening of the Pathogenic Yeast Candida glabrata
1 Introduction
1.1 RNA Interference, Gene Silencing Pathway of Eukaryotes
1.2 Candida glabrata Opportunistic Human Pathogen
1.3 RNAi as a Tool, Applications in Yeast
1.4 Outlook
2 Materials
2.1 Expression of RNAi and Silencing Constructs in C. glabrata
2.2 Gene Library Construction
2.3 Screening Using Robotics
2.4 Macrophages Differentiation
3 Methods
3.1 RNAi, Hairpin, and Antisense Constructs for Gene Silencing in C. glabrata
3.2 Gene Library Construction
3.3 Phenotypic Profiling Using Robotics
3.4 Macrophages
4 Notes
References
Chapter 19: High-Throughput Gene Mutagenesis Screening Using Base Editing
1 Introduction
2 Materials
2.1 gRNA Library Creation (Computational)
2.2 gRNA Library Cloning
2.3 Prepare Yeast Competent Cells with the Target-AID Base Editor
2.4 Library Transformation in Yeast
2.5 Large-Scale Screen
2.6 Sequencing Library Preparation
3 Methods
3.1 gRNA Library Creation (Computational)
3.2 gRNA Library Cloning
3.3 Yeast Competent Cell Preparation with the Target-AID Base Editor
3.4 Library Transformation in in Yeast
3.5 Large-Scale Screen
3.6 Sequencing Library Preparation
4 Notes
References
Chapter 20: SAturated Transposon Analysis in Yeast (SATAY) for Deep Functional Mapping of Yeast Genomes
1 Introduction
2 Materials
2.1 Equipment
2.2 Growth Media
2.3 DNA Work
2.4 Yeast Work
2.5 Sequencing
3 Methods
3.1 Generate Starting Yeast Clones
3.1.1 Verify pBK549 by Restriction Digest
3.1.2 Transform pBK549 in Strains of Interest
3.1.3 Selection of Yeast Transformants
3.2 Transposition
3.2.1 Day 0
3.2.2 Day 1
3.2.3 Day 4
3.2.4 Days 5-20
3.3 Plate Harvest, Library Expansion, and Collection
3.3.1 Harvest the Plates
3.3.2 Reseed
3.3.3 Final Harvest
3.4 DNA Template Preparation
3.4.1 Genomic DNA Extraction
3.4.2 Genomic DNA Quantification on Gel (See Note 39)
3.4.3 Genomic DNA Digestion with NlaIII and DpnII
3.4.4 Digested gDNA Circularization
3.5 Library Amplification
3.5.1 Primer Design
3.5.2 PCR and Purification
3.6 Sequencing
3.6.1 Combine Libraries to Be Sequenced Together
3.6.2 Library Preparation
3.6.3 Loading of Library
3.6.4 Loading of Sequencing Primer (Sequencing of the Transposon-Genome Junctions)
3.6.5 Loading of Custom Index Primer (Sequencing of Indexes)
3.6.6 Instrument Setup
3.7 Analysis
3.7.1 Reads Mapping Using R Script
3.7.2 Reads Mapping Using Web Server
3.7.3 Visualizing Transposon Maps
4 Notes
References
Chapter 21: High-Throughput, High-Precision Colony Phenotyping with Pyphe
1 Introduction
2 Materials
2.1 Experimental
2.2 Scanning and Data Processing
3 Methods
3.1 General Method: Endpoints
3.1.1 Experimental Design and Plating
3.1.2 Acquisition
3.1.3 Quantification
3.1.4 Analysis
3.1.5 Interpretation
3.2 Variation 1: Time-Course Analysis
3.3 Variation 2: Colony Viability Analysis
4 Notes
References
Chapter 22: Bulk-Fitness Measurements Using Barcode Sequencing Analysis in Yeast
1 Introduction
2 Materials
2.1 Yeast Genomic DNA Extraction Using Silica Mini-Preparative Columns
2.2 Two-Step PCR
2.3 Growth Media
2.4 Softwares
3 Methods
3.1 Growth Assays
3.2 Genomic Extractions of Yeasts
3.3 Two-Step PCR Amplification of Barcodes
3.4 Parsing Sequencing Reads into Barcodes
3.5 Fitness Inference
4 Notes
References
Part V: In Silico Integration of Functional Genomics Data
Chapter 23: Prediction of Gene and Genomic Regulation in Candida Species, Using the PathoYeastract Database: A Comparative Gen...
1 Introduction
2 Methods
2.1 Predicting the Regulation of Individual Genes Based on Data for the Species under Study
2.2 Predicting the Regulation of Individual Genes Based on Data for Other Species in YEASTRACT+
2.3 Predicting the Targets of Individual TFs Based on Data for the Species Under Study
2.4 Predicting the Targets of Individual TFs Based on Data for Other Species in YEASTRACT+
2.5 Transcriptomics Data Analysis Based on Documented Data of Regulatory Interactions for the Species Under Study
2.6 Transcriptomics Data Analysis Based on Data for Other Species in PathoYeastract
3 Notes
References
Chapter 24: Enabling Studies of Genome-Scale Regulatory Network Evolution in Large Phylogenies with MRTLE
1 Introduction
2 Materials
3 Methods
3.1 The Theory of MRTLE Model
3.2 Downloading and Installing MRTLE
3.3 Preparing Inputs for MRTLE
3.3.1 Configuration File
3.3.2 Expression Data File
3.3.3 Species-Specific Regulators File
3.3.4 Species-Specific Targets File
3.3.5 Motif-Based Prior Network File (Optional)
3.3.6 Tree File
3.3.7 Gene Orthology Mappings File (OGID File)
3.3.8 Species List File
3.3.9 Potential Regulator Orthogroups ID File
3.3.10 Potential Target Orthogroups ID File
3.4 Applying MRTLE to Multi-Species Data
3.5 MRTLE Outputs and their Interpretation
4 Notes
References
Chapter 25: Omics Analyses: How to Navigate Through a Constant Data Deluge
1 Introduction
2 Basics for Data Analysis
2.1 Basics #1: Differentiate Data,´´Information,´´ and ``Knowledge´´
2.2 Basics #2: Recognize the Importance of Data Visualization
2.3 Basics #3: Think of Data Analysis as a Cycle
2.3.1 Formulation of the Scientific Question
2.3.2 Collection (and Cleaning) of Available Data
2.3.3 Data Exploration and Preliminary Analyses
2.3.4 Formulation of Statistical Hypotheses
2.3.5 Interpretation and Conclusions
3 Specific Statements in Omics Data Analyses
3.1 Be Aware of Cherry Picking
3.2 Be Aware of P-Hacking
4 Our Ten Recommended Rules
5 Conclusion
References
Index