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Related Concept Videos

RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...

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Related Experiment Video

Updated: May 29, 2026

Identification of Circular RNAs using RNA Sequencing
08:25

Identification of Circular RNAs using RNA Sequencing

Published on: November 14, 2019

A strand-specific library preparation protocol for RNA sequencing.

Tatiana Borodina1, James Adjaye, Marc Sultan

  • 1Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Ihnestrasse 63-73, Berlin, Germany.

Methods in Enzymology
|September 28, 2011
PubMed
Summary
This summary is machine-generated.

Next-generation sequencing (NGS) offers advanced transcriptome analysis. This study details a deoxy-UTP (dUTP) strand-marking protocol for strand-specific RNA sequencing (RNA-Seq) library preparation.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Microarray technology has been the primary method for transcriptome analysis.
  • Next-generation sequencing (NGS) systems are now superseding microarrays.
  • RNA sequencing (RNA-Seq) is a powerful NGS application for gene expression and transcript structure analysis.

Purpose of the Study:

  • To describe a step-by-step deoxy-UTP (dUTP) strand-marking protocol.
  • To provide a leading protocol for strand-specific RNA-Seq library preparation.
  • To enable detailed analysis of splicing events, RNA editing, and mutations.

Main Methods:

  • Isolation and fragmentation of polyA fraction (mRNA) from total RNA.
  • Double-stranded (ds) cDNA synthesis incorporating dUTP in the second strand.
  • Standard Illumina sequencing library preparation with selective dUTP-marked strand degradation using Uracil-DNA-Glycosylase (UDG) prior to amplification.

Main Results:

  • The dUTP strand-marking protocol allows for strand-specific RNA-Seq library preparation.
  • The protocol integrates seamlessly with standard Illumina sequencing workflows.
  • Selective degradation of the dUTP-marked strand ensures accurate sequencing of the original RNA strand.

Conclusions:

  • The described dUTP strand-marking protocol is a leading method for strand-specific RNA-Seq.
  • This technique enhances the depth of transcriptome analysis.
  • It provides crucial information on gene expression, transcript structure, and sequence variations.