Kooperativer Bibliotheksverbund

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Format: 1 online resource (445 pages)

Edition: 2nd ed.

ISBN: 1-4939-9736-X

Series Statement: Methods in Molecular Biology ; v.2049

Note: Intro -- Dedication -- Author Biography -- Preface -- Contents -- Contributors -- Part I: Yeast Systems Biology -- Chapter 1: Yeast Systems Biology: The Continuing Challenge of Eukaryotic Complexity -- 1 Introduction -- 2 Advances in `Omic Technologies -- 3 Multiple Levels of Control -- 4 The Rise of Mathematical Modeling and the Integration of Data from the Different `Omic Domains and Timescales -- 5 The Relationship Between Systems and Synthetic Biology and New Biological Workbenches -- 6 Yeast as a Model for Both Infectious and Systemic Human Diseases -- 7 Conclusions and Prospects -- References -- Part II: Experimental Systems Biology: High-Throughput Genome-Wide and Molecular Studies -- Chapter 2: Capture of Ribonucleotides in Yeast Genomic DNA Using Ribose-Seq -- 1 Introduction -- 2 Materials -- 2.1 Yeast Genomic DNA Extraction -- 2.2 Annealing of Ribose-Seq Adaptor Oligonucleotides -- 2.3 Ribose-Seq -- 2.3.1 Fragmentation of DNA -- 2.3.2 dA-Tailing and Adaptor Ligation -- 2.3.3 Alkali Treatment -- 2.3.4 AtRNL Ligation -- 2.3.5 T5 Exonuclease Degradation -- 2.3.6 Tpt1 Reaction -- 2.3.7 PCR Amplification of DNA Libraries -- 2.3.8 PAGE Visualization -- 2.4 Equipment -- 3 Methods -- 3.1 Yeast Genomic DNA Extraction -- 3.2 Preparation of Ribose-Seq Adaptor -- 3.3 Ribose-Seq -- 3.3.1 Fragmentation of Genomic DNA -- 3.3.2 dA-Tailing and Adaptor Ligation -- 3.3.3 Alkali Treatment -- 3.3.4 Self-Ligation (Circularization) of 2'-Phosphate Terminal of rNMP to 5'-Phosphate of the DNA by AtRNL -- 3.3.5 Removal of Linear ssDNA -- 3.3.6 Removal of 2'-Phosphate -- 3.4 PCR -- 3.4.1 PCR 1 -- 3.4.2 PCR 2 -- 3.5 PAGE -- 3.6 Size Selection and Gel Purification -- 4 Notes -- References -- Chapter 3: Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9 -- 1 Introduction -- 2 Materials -- 2.1 Cas9 Yeast Strain. , 2.2 DNA Constructs and Templates -- 2.3 Primers -- 2.4 Lithium Acetate Transformation -- 2.5 Yeast Media -- 3 Methods -- 3.1 Preparation of Cas9-Expressing Base Strain -- 3.2 Colony PCR Confirmation of the Cas9 Base Strain -- 3.3 CRISPR Target Site Selection for Deletion of an Open Reading Frame or Region -- 3.4 CRISPR Target Site Selection for Integration of a Point Mutation -- 3.5 Cloning-Free Generation of Guide RNA Constructs -- 3.6 Generation of Linear Entry Vector -- 3.7 Donor DNA Design -- 3.8 Generation of Short Donor DNAs (Including the JEN1 Control Donor) by Primer Extension -- 3.9 Markerless, Multiplex Engineering in the Cas9 Base Strain -- 4 Notes -- Appendix: Primers and Sequences -- Genbank Sequence: pAH1 gRNA Expression Plasmid -- Genbank Sequence: pAH2 Cas9 Expression Construct 1 of 2 (PmeI Fragment) -- Genbank Sequence: pAH3 Cas9 Expression Construct 2 of 2 (PmeI Fragment) -- Genbank Sequence: pAH2 Recombined with pAH3 at Integration Site -- References -- Chapter 4: Ultrahigh-Density Screens for Genome-Wide Yeast EMAPs in a Single Plate -- 1 Introduction -- 2 Materials -- 2.1 Strains and Plasmids for Generation of Query Strains -- 2.2 Drug Concentrations -- 2.3 Transformation Media and Solutions -- 2.4 Transformation Media for DAmP Essential Strains -- 2.5 Synthetic Genetic Array (SGA) Screen -- 2.5.1 Strains -- 2.5.2 Media -- 3 Methods -- 3.1 Designing Transformation Products -- 3.2 Making Yeast Query Competent Cells -- 3.3 Cell Transformation for Essential and Nonessential Genes -- 3.4 Validate Strains (Colony PCR) -- 3.5 Yeast DAmP Protocol for Making Essential Strains -- 3.6 Synthetic Genetic Array (SGA) -- 3.6.1 Preparing Query Lawn -- 3.6.2 Preparing Library (See Note 12) -- 3.6.3 Mating -- 3.6.4 Diploid Selection -- 3.6.5 Sporulation -- 3.6.6 Haploid Selection 1 -- 3.6.7 Haploid Selection 2 -- 3.6.8 Single Mutant Selection. , 3.6.9 Double Mutant Selection -- 3.7 Digital Imaging -- 3.8 Data Analysis -- 3.8.1 Image Processing and Spot Size Detection -- 3.8.2 Compute S-Scores -- 4 Notes -- References -- Chapter 5: Epi-ID: Systematic and Direct Screening for Chromatin Regulators in Yeast by Barcode-ChIP-Seq -- 1 Introduction -- 2 Materials -- 2.1 Yeast Strains and Plasmids -- 2.2 Yeast Media and Drugs -- 2.3 Yeast Plates and Accessories -- 2.4 Buffers -- 2.5 Equipment -- 3 Methods -- 3.1 Cell Culture and Preparation of Cell Pellets -- 3.2 Coupling Beads and Antibody -- 3.3 Chromatin Preparation -- 3.3.1 Preparing 15 ml Bioruptor Pico Tubes for Sonication -- 3.3.2 Preparing Chromatin -- 3.3.3 Check the Chromatin Size -- 3.4 Chromatin Immunoprecipitation -- 3.5 Library Preparation -- 3.5.1 Test PCR Analysis for ChIP Efficiency -- 3.5.2 Library Preparation -- 3.6 Sequencing and Data Analysis -- 4 Notes -- References -- Chapter 6: Transcript Profiling Analysis Through Paired-End Ditag (PET) Approach Coupled with Deep Sequencing Reveals Transcri... -- 1 Introduction -- 2 Materials -- 2.1 Strain, Growth Conditions -- 2.2 M280 Beads Buffer -- 3 Methods -- 3.1 RNA Extraction and mRNA Isolation -- 3.2 PET Library Preparation -- 3.2.1 10 μg mRNA Is Reversely Transcribed (RT) into cDNA with GsuI-Oligo dT Primer (Table 1) as Below -- 3.2.2 Full-Length cDNA Selection -- 3.2.3 Rolling Circle Amplification (RCA) of Circular Full-Length DNA Molecules -- 3.2.4 PET Fragment Generation -- 3.3 Data Processing -- 3.4 Uncharacterized Transcript Validation -- 3.5 Rapid Amplification of cDNA Ends (RACE) Validation -- 3.6 Gene Ontology Analysis -- 4 Notes -- References -- Chapter 7: RNA Sequencing Best Practices: Experimental Protocol and Data Analysis -- 1 Introduction -- 2 Materials -- 2.1 Experimental Design -- 2.2 RNA Isolation and Quality Control (See Note 1). , 2.3 RNA-Seq Read Processing (See Note 3) -- 2.4 Downstream Analysis of RNA-Seq Count Data (See Note 6) -- 3 Methods -- 3.1 Experimental Design -- 3.2 RNA Isolation and Quality Control (See Note 8) -- 3.3 RNA-Sequencing -- 3.4 RNA-Seq Read Processing -- 3.5 Downstream Analysis of RNA-Seq Count Data -- 3.6 Database Submission -- 4 Notes -- References -- Chapter 8: Advanced Methods for the Analysis of Altered Pre-mRNA Splicing in Yeast and Disease -- 1 Introduction -- 2 Materials -- 2.1 Yeast Culture -- 2.2 RNA Extraction -- 2.3 Bioinformatic Analysis (Software and Reference Files) -- 2.4 cDNA Synthesis -- 2.5 PCR Amplification -- 2.6 Agarose Gel Electrophoresis -- 3 Methods -- 3.1 Yeast Culture -- 3.2 RNA Isolation and Preparation -- 3.3 Bioinformatic Analyses -- 3.4 Data Validation by RT-PCR -- 4 Notes -- References -- Chapter 9: PAT-Seq: A Method for Simultaneous Quantitation of Gene Expression, Poly(A)-Site Selection and Poly(A)-Length Distr... -- 1 Introduction -- 2 Materials -- 2.1 End Extension of Adenylated RNA to Generate the 3'-Tag -- 2.2 Limited Fragmentation of RNA with RNaseT1 -- 2.3 Enrichment of 3' Fragments and Their 5' Phosphorylation -- 2.4 Ligation of 5'-Splint-Linker to the 5'-Ends of 3' Fragments -- 2.5 Reverse Transcription -- 2.6 Size Selection of PAT-Seq Library -- 2.7 Barcoded PAT-Seq Library Amplification -- 2.8 Facility for Illumina-Based NGS -- 3 Methods -- 3.1 End-Extension of Adenylated RNA to Generate the 3'-Tag -- 3.2 Limited Fragmentation of RNA with RNaseT1 -- 3.3 Enrichment of 3' Fragments and Their 5' Phosphorylation -- 3.4 Phosphorylation of 5'-Ends of RNA Fragments -- 3.5 Ligation of 5'-Splint-Adapter to the 5'-Ends of 3'Fragments -- 3.6 Reverse Transcription -- 3.7 Urea-PAGE Size Selection of PAT-Seq Library -- 3.8 PCR Amplification and Indexing of the PAT-Seq Library -- 3.9 Sequencing of PAT-Seq Libraries. , 3.10 Concluding Remarks -- 4 Notes -- References -- Chapter 10: Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT -- 1 Introduction -- 2 Materials and Equipment -- 2.1 Cell Culture and Harvest -- 2.2 Cell Pretreatment and Lysis -- 2.3 Ultracentrifugation -- 2.4 Protein Fraction Processing and Concentration Estimation -- 2.5 Digestion, TMT Labeling, Peptide Desalting, and Prefractionation -- 3 Methods -- 3.1 Cell Culture -- 3.2 Cell Harvest and Preprocessing -- 3.3 Nuclear Preparation (Optional) -- 3.4 Spheroplast Lysis for Density Gradient Fractionation -- 3.5 Subcellular Fractionation -- 3.6 Fraction Processing -- 3.7 Tryptic Digestion -- 3.8 TMT Labeling -- 3.9 Sample Cleanup by Solid-Phase Extraction on Sep Pak Reversed Phase Cartridges -- 3.10 Off-Line High pH Reversed Phase Fractionation -- 3.11 Mass Spectrometry Data Processing -- 3.12 Analysis of Spatial Proteomics Data, Protein Classification and Assignment -- 4 Notes -- References -- Chapter 11: Data-Independent Acquisition for Yeast Glycoproteomics -- 1 Introduction -- 1.1 Mass Spectrometry Proteomics -- 1.2 N-Glycoproteins and Their Relevance in Physiology -- 1.3 Diversity in N-Glycosylation -- 1.4 Glycoproteomics as a Tool to Study Protein Glycosylation -- 2 Materials -- 2.1 Cell Growth and Harvest -- 2.2 Cell Lysis and Preparation for MS Analysis -- 2.3 Solutions for Cell Lysis and Preparation for MS Analysis (See Note 1) -- 2.4 Zip-Tipping with C18 ZipTips -- 2.5 Solutions for Zip-Tipping with a C18 ZipTip -- 3 Methods -- 3.1 Yeast Growth and Harvest -- 3.2 Cell Wall Preparation -- 3.3 Deglycosylation of Cell Wall Protein Extracts (See Note 16) -- 3.4 ZipTipping Protocol for C18 Ziptips (See Notes 18 and 19) -- 3.5 Mass Spectrometry -- 3.5.1 Analyze Peptides and Glycopeptides with Data Dependent Acquisition Mass Spectrometry. , 3.5.2 Analyze Peptides and Glycopeptides with Data-Independent Acquisition (SWATH) Mass Spectrometry.

Additional Edition: ISBN 1-4939-9735-1

Language: English