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Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

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Illuminating Enhancer Transcription at Nucleotide Resolution with Native Elongating Transcript Sequencing (NET-Seq).

Olga Jasnovidova¹, Mirjam Arnold^1,2, Andreas Mayer³

¹Otto-Warburg-Laboratory, Max Planck Institute for Molecular Genetics, Berlin, Germany.

Methods in Molecular Biology (Clifton, N.J.)

|August 12, 2021

Summary

This study details native elongating transcript sequencing (NET-Seq) for precise in vivo measurement of enhancer transcription. This method enables accurate tracking of RNA polymerase II activity genome-wide.

Keywords:

Enhancer Genome-wide Library preparation NET-Seq Nascent RNA Next-generation sequencing RNA polymerase II Transcription eRNA

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Area of Science:

Molecular Biology
Genomics
Epigenetics

Background:

Enhancers regulate target gene expression through transcription.
Understanding enhancer transcription is crucial for gene control.
Existing methods lack the precision to study enhancer transcription in vivo.

Purpose of the Study:

To provide a detailed protocol for Native Elongating Transcript Sequencing (NET-Seq) in mammalian cells.
To enable precise, quantitative, and genome-wide measurement of enhancer transcription.
To facilitate the study of enhancer transcription regulation and its role in target gene control.

Main Methods:

Step-by-step guidance for performing NET-Seq in mammalian cells.
Capturing and converting 3'-ends of nascent RNA into sequencing libraries.
Next-generation sequencing and computational analysis of transcription data.

Main Results:

NET-Seq provides quantitative measurements of transcription genome-wide.
The method offers single-nucleotide and DNA strand resolution.
A NET-Seq library can be obtained within 5 days, including quality control.

Conclusions:

NET-Seq is a powerful tool for studying enhancer transcription in vivo.
The protocol allows for precise monitoring of RNA polymerase II activity at enhancers.
This method advances the understanding of gene regulation by enhancers.