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Illuminating enhancer transcription at nucleotide resolution with native elongating transcript sequencing (NET-Seq)

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Jasnovidova,  Olga
High-Resolution Functional Genomics (Andreas Mayer), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Arnold,  Mirjam
High-Resolution Functional Genomics (Andreas Mayer), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Mayer,  Andreas
High-Resolution Functional Genomics (Andreas Mayer), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Citation

Jasnovidova, O., Arnold, M., & Mayer, A. (2021). Illuminating enhancer transcription at nucleotide resolution with native elongating transcript sequencing (NET-Seq). In T. Borggrefe (Ed.), Enhancers and Promoters - Methods and Protocols (Methods in Molecular Biology; 2351) (pp. 41-65). New York, NY: Humana. doi:0.1007/978-1-0716-1597-3_3.


Cite as: https://hdl.handle.net/21.11116/0000-0009-3E64-2
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.