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Related Concept Videos

RNA-seq03:21

RNA-seq

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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. 
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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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Next-Generation Sequencing RNA-Seq Library Construction.

Jessica Podnar1, Heather Deiderick1, Gabriella Huerta1

  • 1Genomic Sequencing and Analysis Facility, University of Texas at Austin, Austin, Texas.

Current Protocols in Molecular Biology
|April 16, 2014
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Summary

This study details robust protocols for creating directional RNA sequencing libraries for next-generation sequencing (NGS) on Illumina platforms. These methods preserve RNA orientation for strand-specific analysis, suitable for various applications and high-throughput labs.

Keywords:
NGSRNA-Seqgene expressionlibrary constructionstrand-specifictranscriptome

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Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Next-generation sequencing (NGS) is crucial for transcriptome analysis.
  • Strand-specific RNA sequencing provides more accurate gene expression data.
  • Efficient library preparation protocols are essential for high-throughput studies.

Purpose of the Study:

  • To present robust protocols for constructing directional RNA sequencing libraries for Illumina platforms (HiSeq and MiSeq).
  • To enable strand-specific analysis of RNA sequencing data by preserving original RNA orientation.
  • To provide a versatile protocol applicable to diverse RNA input types and qualities for high-throughput laboratories.

Main Methods:

  • Utilized protocols based on the New England Biolabs (NEB) small RNA library preparation set for Illumina.
  • Adapted protocols for various RNA input sources, ensuring preservation of RNA orientation.
  • Implemented library preparation for directional RNA sequencing.

Main Results:

  • Developed and validated protocols for directional RNA sequencing library construction.
  • Demonstrated preservation of RNA orientation, enabling strand-specific analysis.
  • Established a robust protocol applicable to a wide range of RNA input types and qualities.

Conclusions:

  • The presented protocols are effective for generating directional RNA sequencing libraries for Illumina platforms.
  • The method is suitable for differential gene expression analysis, small RNA discovery, and de novo transcriptome assembly.
  • The protocol's robustness and versatility make it ideal for high-throughput genomics laboratories.