Enzymes in RNA science and biotechnology. Part B /

Enzymes in RNA Science and Biotechnology, Part B, Volume 692 in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters on topics such as Quantitative base-resolution sequencing technology for mapping pseudouridines in mammalian mRN...

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Detalles Bibliográficos
Clasificación:Libro Electrónico
Otros Autores: Yamagami, Ryota (Editor ), Kwok, Chun Kit (Editor )
Formato: Electrónico eBook
Idioma:Inglés
Publicado: Amsterdam, Netherlands : Elsevier Academic Press, 2023.
Edición:First edition.
Colección:Methods in enzymology, volume 692
Temas:
Catalytic RNA.
Enzymes > Biotechnology.
Ribozymes.
Enzymes > Biotechnologie.
Acceso en línea:Texto completo
Tabla de Contenidos:
  • Front Cover
  • Series Page
  • Methods in ENZYMOLOGY
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Section 1: Post-transcriptional modification enzymes
  • Chapter One: D-Seq: Genome-wide detection of dihydrouridine modifications in RNA
  • 1 Introduction
  • 1.1 RNA modifications impact RNA function
  • 1.2 Methods to detect dihydrouridine
  • 2 Step-by-step method details
  • 2.1 Sample preparation
  • 2.1.1 RNA isolation from S. cerevisiae
  • 2.1.2 Poly(A) selection using oligo (dT) cellulose beads
  • 2.1.3 Poly(A) selection using oligo (dT) magnetic beads
  • 2.1.4 tRNA size selection using silica columns
  • 3 D-Seq Library preparation
  • 3.1 RNA fragmentation
  • 3.2 Borohydride reduction
  • 3.3 32 End healing
  • 3.4 RNA size selection
  • 3.5 32 Adapter ligation
  • 3.6 Reverse transcription
  • 3.7 Size selection
  • 3.8 52 Adapter ligation
  • 3.9 PCR amplification
  • 4 D-seq data analysis
  • 4.1 Trimming of adapter sequences
  • 4.2 PCR duplicate collapsing
  • 4.3 UMI removal
  • 4.4 Read mapping
  • 4.5 Read end position gathering
  • 4.6 D-site identification
  • 5 Experimental considerations
  • 5.1 Negative control selection
  • 5.2 Sequencing depth and biological replicates
  • 6 Solutions and reagent recipes
  • 6.1 Solutions
  • Acknowledgments
  • References
  • Chapter Two: Quantitative base-resolution sequencing technology for mapping pseudouridines in mammalian mRNAQuantitative base-resolution sequencing technology
  • 1 Introduction
  • 2 Materials
  • 2.1 Preparation of RNA samples
  • 2.2 RNA fragmentation
  • 2.3 RNA 32- and 52-end repair
  • 2.4 RNA 32-adaptor ligation
  • 2.5 RNA 52-adaptor ligation
  • 2.6 BID-seq bisulfite treatment
  • 2.7 Desulphonation
  • 2.8 Reverse transcription
  • 2.9 PCR amplification
  • 2.10 NGS sequencing and data analysis
  • 3 Methods
  • 3.1 Overview
  • 3.2 Preparation of RNA sample
  • 3.3 RNA fragmentation
  • 3.4 RNA 32- and 52-end repair
  • 3.5 RNA 32-adaptor ligation
  • 3.6 RNA 52 adaptor ligation
  • 3.7 BID-seq bisulfite treatment
  • 3.8 Desulphonation
  • 3.9 Reverse transcription
  • 3.10 PCR amplification
  • 3.11 NGS sequencing and data analysis
  • 4 Notes
  • Funding
  • References
  • Chapter Three: Base-resolution quantitative DAMM-seq for mapping RNA methylations in tRNA and mitochondrial polycistronic RNABase-resolution quantitative DAMM-seq
  • 1 Introduction
  • 2 Materials
  • 2.1 Preparation of RNA samples
  • 2.2 RNA fragmentation
  • 2.3 Demethylation
  • 2.4 RNA 32-end repair
  • 2.5 RNA 32-adaptor ligation
  • 2.6 Reverse transcription
  • 2.7 cDNA 32-adaptor ligation
  • 2.8 PCR amplification
  • 2.9 NGS sequencing and data analysis
  • 3 Methods
  • 3.1 Overview of DAMM-seq
  • 3.2 Preparation of RNA sample
  • 3.3 RNA fragmentation
  • 3.4 Demethylation (optional)
  • 3.5 RNA 32-end repair
  • 3.6 RNA 32-adaptor ligation
  • 3.7 Reverse transcription
  • 3.8 cDNA 32-end ligation
  • 3.9 PCR amplification
  • 3.10 NGS sequencing and data analysis
  • 4 Notes

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