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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
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Global Run-On Sequencing (GRO-Seq).

Alessandro Gardini1

  • 1The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, 19104, USA. agardini@wistar.org.

Methods in Molecular Biology (Clifton, N.J.)
|September 25, 2016
PubMed
Summary

This study introduces a Nuclear Run-On assay with deep sequencing to measure real-time gene transcription. This method accurately quantifies RNA polymerase activity and maps nascent transcripts genome-wide.

Keywords:
Deep sequencingEnhancer RNANascent RNANoncoding RNANuclear run-onRNA polymeraseTranscription elongationTranscription initiation

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Area of Science:

  • Molecular Biology
  • Genomics
  • Transcriptomics

Background:

  • Mature RNA levels (e.g., mRNAs) measured by RT-PCR or RNA sequencing do not precisely reflect transcriptional activity.
  • RNA stability significantly influences the abundance of RNA molecules, complicating direct measurement of transcription.
  • Accurate assessment of real-time transcription is crucial for understanding gene regulation.

Purpose of the Study:

  • To develop and describe a protocol for assessing real-time transcription using Nuclear Run-On assay coupled with deep sequencing.
  • To provide a reliable method for measuring transcriptional activity at the genome-wide scale in mammalian cells.
  • To enable high-resolution mapping and quantification of both coding and noncoding nascent transcripts.

Main Methods:

  • Nuclear Run-On (NRO) assay to capture actively transcribing RNA polymerase.
  • Deep sequencing (NGS) to map nascent transcripts across the genome.
  • Integration of NRO with sequencing for genome-wide transcriptional profiling.

Main Results:

  • The combined NRO-sequencing protocol accurately measures real-time transcriptional activity.
  • Genome-wide mapping of nascent transcripts provides a high-resolution view of transcription.
  • The method is particularly effective for annotating and quantifying short-lived RNA molecules.

Conclusions:

  • Nuclear Run-On assay coupled with deep sequencing offers a robust measure of transcriptional activity.
  • This approach overcomes limitations of steady-state RNA level measurements.
  • The protocol facilitates precise genome-wide transcriptional analysis and RNA annotation.