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  A detailed protocol for subcellular RNA sequencing (subRNA-seq)

Mayer, A., & Churchman, L. S. (2017). A detailed protocol for subcellular RNA sequencing (subRNA-seq). In F. M. Ausubel (Ed.), Current Protocols in Molecular Biology (pp. 4.29.1-4.29.18). Hoboken, NJ: John Wiley & Sons, Inc. doi:10.1002/cpmb.44.

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Mayer.pdf (Publisher version), 441KB
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 Creators:
Mayer, Andreas1, Author           
Churchman, L. Stirling 2, Author
Affiliations:
1Nascent Transcription and Cell Differentiation (Andreas Mayer), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_2385699              
2Department of Genetics, Harvard Medical School, Boston, Massachusetts, ou_persistent22              

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Free keywords: Cell fractionation; RNA polymerase II (Pol II); RNA processing; nascent RNA; next-generation sequencing; subcellular RNA-seq; transcription
 Abstract: In eukaryotic cells, RNAs at various maturation and processing levels are distributed across cellular compartments. The standard approach to determine transcript abundance and identity in vivo is RNA sequencing (RNA-seq). RNA-seq relies on RNA isolation from whole-cell lysates and thus mainly captures fully processed, stable, and more abundant cytoplasmic RNAs over nascent, unstable, and nuclear RNAs. Here, we provide a step-by-step protocol for subcellular RNA-seq (subRNA-seq). subRNA-seq allows the quantitative measurement of RNA polymerase II-generated RNAs from the chromatin, nucleoplasm, and cytoplasm of mammalian cells. This approach relies on cell fractionation prior to RNA isolation and sequencing library preparation. High-throughput sequencing of the subcellular RNAs can then be used to reveal the identity, abundance, and subcellular distribution of transcripts, thus providing insights into RNA processing and maturation. Deep sequencing of the chromatin-associated RNAs further offers the opportunity to study nascent RNAs. Subcellular RNA-seq libraries are obtained within 5 days

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Language(s): eng - English
 Dates: 2017-10-02
 Publication Status: Published online
 Pages: 18
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: DOI: 10.1002/cpmb.44
 Degree: -

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Title: Current Protocols in Molecular Biology
Source Genre: Series
 Creator(s):
Ausubel, Frederick M. 1, Editor
Affiliations:
1 Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts , ou_persistent22            
Publ. Info: Hoboken, NJ : John Wiley & Sons, Inc.
Pages: - Volume / Issue: Suppl. 120 Sequence Number: - Start / End Page: 4.29.1 - 4.29.18 Identifier: ISSN: 1934-3647