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Enhancers and Promoters pp 41–65Cite as

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

Part of the Methods in Molecular Biology book series (MIMB,volume 2351)

Abstract

Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3′-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days.

Keywords

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

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  • DOI: 10.1007/978-1-0716-1597-3_3
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Acknowledgments

We thank all members of the Mayer group for critical comments on the manuscript. This work was funded by the Max Planck Society (to A.M.) and the Deutsche Forschungsgemeinschaft (DFG, grant no. 418415292 to A.M. and the International Research Training Group (IRTG) 2403 to A.M. and M.A.). O.J. was supported by a 2017 FEBS Long-Term Fellowship.

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Author notes

  1. Olga Jasnovidova and Mirjam Arnold contributed equally to this work.

Authors and Affiliations

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

    Olga Jasnovidova, Mirjam Arnold & Andreas Mayer

  2. Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Berlin, Germany

    Mirjam Arnold

Authors
  1. Olga Jasnovidova
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  2. Mirjam Arnold
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  3. Andreas Mayer
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Corresponding author

Correspondence to Andreas Mayer .

Editor information

Editors and Affiliations

  1. Institute of Biochemistry, University of Giessen, Gießen, Hessen, Germany

    Prof. Dr. Tilman Borggrefe

  2. Institute of Biochemistry, University of Giessen, Gießen, Hessen, Germany

    Dr. Benedetto Daniele Giaimo

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Jasnovidova, O., Arnold, M., Mayer, A. (2021). Illuminating Enhancer Transcription at Nucleotide Resolution with Native Elongating Transcript Sequencing (NET-Seq) . In: Borggrefe, T., Giaimo, B.D. (eds) Enhancers and Promoters. Methods in Molecular Biology, vol 2351. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1597-3_3

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  • DOI: https://doi.org/10.1007/978-1-0716-1597-3_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1596-6

  • Online ISBN: 978-1-0716-1597-3

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