mRNA 3’ End Processing and Metabolism

1st Edition, Volume 655 - June 25, 2021
Imprint: Academic Press
Editor: Bin Tian
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 2 3 5 7 3 - 7
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 2 3 5 7 4 - 4

mRNA 3’ End Processing and Stability, Volume 655 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters… Read more

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mRNA 3’ End Processing and Stability, Volume 655 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of timely topics. Each chapter is written by an international board of authors.

Cover image
Title page
Table of Contents
Copyright
Contributors
Preface
Chapter One: Application and design considerations for 3′-end sequencing using click-chemistry
Abstract
1: Introduction
2: Next-generation sequencing tools to detect and measure alternative polyadenylation
3: PolyA-ClickSeq library preparation and sequencing
4: PAC-seq protocol
5: Potential limitations of PAC-seq and considerations for depth
Acknowledgments
Chapter Two: PAS-seq 2: A fast and sensitive method for global profiling of polyadenylated RNAs
Abstract
1: Introduction
2: Materials
3: Methods
4: Data analysis
5: Troubleshooting tips
Acknowledgments
Chapter Three: TRENDseq—A highly multiplexed high throughput RNA 3′ end sequencing for mapping alternative polyadenylation
Abstract
1: Introduction
2: TRENDseq protocol—Overview
3: Bioinformatic pipeline
4: Protocol at glance
5: Summary and outlook
Acknowledgments
Appendix:: ERC protocol
Chapter Four: QPAT-seq, a rapid and deduplicatable method for quantification of poly(A) site usages
Abstract
1: Introduction
2: QPAT-seq library preparation
3: Protocol
4: Discussion
5: Summary
6: Notes
Acknowledgments
Chapter Five: Using TIF-Seq2 to investigate association between 5´ and 3´mRNA ends
Abstract
1: Introduction
2: Protocol
3: Bioinformatic analysis
4: Notes
5: Summary
Acknowledgments
Chapter Six: Single-molecule polyadenylated tail sequencing (SM-PAT-Seq) to measure polyA tail lengths transcriptome-wide
Abstract
1: Introduction
2: Protocol: Overall strategy
3: Materials
4: Protocol
5: Additional comments
Acknowledgments
Chapter Seven: 3′ End sequencing of pA+ and pA− RNAs
Abstract
1: Introduction
2: Method overview
3: Protocol
4: Summary
Chapter Eight: Comprehensive profiling of mRNA polyadenylation in specific cell types in vivo by cTag-PAPERCLIP
Abstract
1: Introduction
2: Materials
3: Methods
4: Notes
Acknowledgements
Chapter Nine: A computational pipeline to infer alternative poly-adenylation from 3′ sequencing data
Abstract
1: Introduction
2: Alternative polyadenylation
3: PolyA-miner
4: Alternative polyadenylation analysis using PolyA-miner
5: Impact of sequencing depth on the number of APA changes detected
6: Summary
Acknowledgments
Chapter Ten: Systematic refinement of gene annotations by parsing mRNA 3′ end sequencing datasets
Abstract
1: Introduction
2: Advantages and limitations of 3′ mRNA sequencing approaches
3: Implementation of 3′GAmES
4: Application of 3′GAmES and expected results
5: Alternative approaches
6: Conclusion
Acknowledgments
Chapter Eleven: Computational analysis of alternative polyadenylation from standard RNA-seq and single-cell RNA-seq data
Abstract
1: Introduction
2: Current bioinformatic tools for analyzing APA in RNA-seq data
3: The DaPars algorithm
4: APA analysis in single cells
5: Summary
Chapter Twelve: Quantifying alternative polyadenylation in RNAseq data with LABRAT
Abstract
1: Introduction
2: How LABRAT works
3: Quantifying alternative polyadenylation with LABRAT
4: Quantification of APA in single cell RNAseq data
Acknowledgments
Chapter Thirteen: Poly(A) tail dynamics: Measuring polyadenylation, deadenylation and poly(A) tail length
Abstract
1: Introduction
2: Equipment
3: Chemicals
4: Cleavage and polyadenylation
5: Deadenylation
6: Poly(A) tail length measurements
7: Summary
Acknowledgment
Chapter Fourteen: Reconstitution and biochemical assays of an active human histone pre-mRNA 3′-end processing machinery
Abstract
1: Introduction
2: Preparation of nuclear extracts for histone pre-mRNA 3′-end processing
3: Reconstitution of an active human histone pre-mRNA 3′-end processing machinery
4: Histone pre-mRNA 3′-end processing assays using radio-labeled substrate
5: Histone pre-mRNA 3′-end processing assays using fluorescently labeled substrate
6: Summary
Acknowledgments
Chapter Fifteen: Comprehensive RNP profiling in cells identifies U1 snRNP complexes with cleavage and polyadenylation factors active in telescripting
Abstract
1: Introduction
2: Methods
3: Summary
4: Key resources table
5: Lead contact for reagent and resource sharing
Acknowledgments
Chapter Sixteen: Simultaneous studies of gene expression and alternative polyadenylation in primary human immune cells
Abstract
1: Introduction
2: Overview of the method
3: Detailed protocol
4: Key resources table
5: Materials, reagents and equipment
6: Step-by-step method details
7: Concluding remarks
8: Safety considerations
9: Expected outcomes
10: Quantification and statistical analysis
11: Advantages
12: Limitations
13: Optimization and troubleshooting
Ethical statement
Acknowledgments
Author contributions
Chapter Seventeen: RIPiT-Seq: A tandem immunoprecipitation approach to reveal global binding landscape of multisubunit ribonucleoproteins
Abstract
1: Introduction
2: Before you begin
3: Materials and equipment
4: Step-by-step method details
5: Expected outcomes
6: Quantification and statistical analysis
7: Advantages
8: Limitations
9: Alternative methods/procedures
Chapter Eighteen: Generation of 3′UTR knockout cell lines by CRISPR/Cas9-mediated genome editing
Abstract
1: Introduction
2: Experimental design
3: Protocol
4: Related techniques
Acknowledgments
Chapter Nineteen: Modulation of alternative cleavage and polyadenylation events by dCas9-mediated CRISPRpas
Abstract
1: Introduction
2: Experimental design
3: Materials
4: Methods
5: Discussion
6: Summary
Acknowledgments

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