title: Isolation of the protein and RNA content of active sites of transcription from mammalian cells.
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.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
authors.forename: Svitlana
authors.initials: S
authors.lastname: Melnik
authors.affiliation: German Cancer Research Center (DKFZ) and BioQuant, Heidelberg, Germany.
authors.forename: Maïwen
authors.initials: M
authors.lastname: Caudron-Herger
authors.affiliation: Center for Molecular Medicine, University of Cologne, Cologne, Germany.
authors.forename: Lilija
authors.initials: L
authors.lastname: Brant
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Carr
authors.affiliation: German Cancer Research Center (DKFZ) and BioQuant, Heidelberg, Germany.
authors.forename: Karsten
authors.identifier: http://orcid.org/0000-0001-9951-9395
authors.initials: K
authors.lastname: Rippe
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Cook
authors.affiliation: Center for Molecular Medicine, University of Cologne, Cologne, Germany.
authors.forename: Argyris
authors.initials: A
authors.lastname: Papantonis
mesh_terms: D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics*; D003412:Cricetulus; D020869:Gene Expression Profiling; D006367:HeLa Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D013058:Mass Spectrometry; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D059467:Transcriptome
chemical_list: D011506:Proteins; D012313:RNA