Universität zu Köln

Melnik, Svitlana, Caudron-Herger, Maiwen ORCID: 0000-0002-7030-8153, Brant, Lilija, Carr, Ian M., Rippe, Karsten ORCID: 0000-0001-9951-9395, Cook, Peter R. and Papantonis, Argyris ORCID: 0000-0001-7551-1073 (2016). Isolation of the protein and RNA content of active sites of transcription from mammalian cells. Nat. Protoc., 11 (3). S. 553 - 566. LONDON: NATURE PUBLISHING GROUP. ISSN 1750-2799

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Abstract

Mammalian cell nuclei contain three RNA polymerases (RNA 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Melnik, Svitlana UNSPECIFIED UNSPECIFIED UNSPECIFIED Caudron-Herger, Maiwen UNSPECIFIED orcid.org/0000-0002-7030-8153 UNSPECIFIED Brant, Lilija UNSPECIFIED UNSPECIFIED UNSPECIFIED Carr, Ian M. UNSPECIFIED UNSPECIFIED UNSPECIFIED Rippe, Karsten UNSPECIFIED orcid.org/0000-0001-9951-9395 UNSPECIFIED Cook, Peter R. UNSPECIFIED UNSPECIFIED UNSPECIFIED Papantonis, Argyris UNSPECIFIED orcid.org/0000-0001-7551-1073 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-283977
DOI:	10.1038/nprot.2016.032
Journal or Publication Title:	Nat. Protoc.
Volume:	11
Number:	3
Page Range:	S. 553 - 566
Date:	2016
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
ISSN:	1750-2799
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language NASCENT TRANSCRIPTION; GENE-EXPRESSION; POLYMERASES; DYNAMICS Multiple languages Biochemical Research Methods Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/28397