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:
CreatorsEmailORCIDORCID Put Code
Melnik, SvitlanaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Caudron-Herger, MaiwenUNSPECIFIEDorcid.org/0000-0002-7030-8153UNSPECIFIED
Brant, LilijaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Carr, Ian M.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Rippe, KarstenUNSPECIFIEDorcid.org/0000-0001-9951-9395UNSPECIFIED
Cook, Peter R.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Papantonis, ArgyrisUNSPECIFIEDorcid.org/0000-0001-7551-1073UNSPECIFIED
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:
KeywordsLanguage
NASCENT TRANSCRIPTION; GENE-EXPRESSION; POLYMERASES; DYNAMICSMultiple languages
Biochemical Research MethodsMultiple languages
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/28397

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