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While mapping total and poly-adenylated human transcriptomes has now become routine, characterizing nascent transcripts remains challenging, largely because nascent RNAs have such short half-lives. Here, we describe a simple, fast and cost-effective method to isolate RNA associated with transcription factories, the sites responsible for the majority of nuclear transcription. Following stimulation of human endothelial cells with the pro-inflammatory cytokine TNFα, we isolate and analyse the RNA content of factories by sequencing. Comparison with total, poly(A)(+) and chromatin RNA fractions reveals that sequencing of purified factory RNA maps the complete nascent transcriptome; it is rich in intronic unprocessed transcript, as well as long intergenic non-coding (lincRNAs) and enhancer-associated RNAs (eRNAs), micro-RNA precursors and repeat-derived RNAs. Hence, we verify that transcription factories produce most nascent RNA and confer a regulatory role via their association with a set of specifically-retained non-coding transcripts.

Original publication

DOI

10.1093/nar/gkv390

Type

Journal article

Journal

Nucleic Acids Res

Publication Date

18/08/2015

Volume

43

Keywords

Cells, Cultured, Chromatin, Gene Expression Profiling, Humans, Introns, RNA, RNA Processing, Post-Transcriptional, RNA, Long Noncoding, RNA, Small Untranslated, RNA, Untranslated, Sequence Analysis, RNA, Transcriptome