Melnik, S, Caudron-Herger, M, Brant, L et al. (4 more authors) (2016) Isolation of the protein and RNA content of active sites of transcription from mammalian cells. Nature Protocols, 11 (3). pp. 553-565. ISSN 1754-2189
Abstract
Mammalian cell nuclei contain three RNA polymerases (RNAP I, RNAP II and RNAP III), which transcribe different gene subsets, and whose active forms are contained in supramolecular complexes known as 'transcription factories.' These complexes are difficult to isolate because they are embedded in the 3D structure of the nucleus. Factories exchange components with the soluble nucleoplasmic pool over time as gene expression programs change during development or disease. Analysis of their content can provide information on the nascent transcriptome and its regulators. Here we describe a protocol for the isolation of large factory fragments under isotonic salt concentrations in <72 h. It relies on DNase I-mediated detachment of chromatin from the nuclear substructure of freshly isolated, unfixed cells, followed by caspase treatment to release multi-megadalton factory complexes. These complexes retain transcriptional activity, and isolation of their contents is compatible with downstream analyses by mass spectrometry (MS) or RNA-sequencing (RNA-seq) to catalog the proteins and RNA associated with sites of active transcription.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2016, Nature America Inc. This is an author produced version of a paper published in Nature Protocols. Uploaded in accordance with the publisher's self-archiving policy. |
Dates: |
|
Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Inst of Biomed & Clin Sciences (LIBACS) (Leeds) > Genetics (LIBACS) (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 09 Mar 2016 10:47 |
Last Modified: | 16 Nov 2016 08:03 |
Published Version: | http://dx.doi.org/10.1038/nprot.2016.032 |
Status: | Published |
Publisher: | Nature Publishing Group |
Identification Number: | 10.1038/nprot.2016.032 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:96057 |