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Methods. 2014 May 1;67(1):45-54. doi: 10.1016/j.ymeth.2013.08.015. Epub 2013 Aug 21.

Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA.

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  • 1Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA.
  • 2Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA; Bioinformatics Program, University of Michigan, Ann Arbor, MI, USA.
  • 3Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA; Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI, USA.
  • 4Department of Pathology, University of Michigan, Ann Arbor, MI, USA; Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA.
  • 5Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center and Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA; Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI, USA. Electronic address: ljungman@umich.edu.

Abstract

Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in the steady-state reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.

Copyright © 2013 Elsevier Inc. All rights reserved.

KEYWORDS:

RNA splicing; RNA stability; Transcription

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