Unit

UNIT 4.18 Genome-Wide Annotation and Quantitation of Translation by Ribosome Profiling

  1. Nicholas T. Ingolia1,2,3,
  2. Gloria A. Brar1,2,
  3. Silvia Rouskin1,2,
  4. Anna M. McGeachy3,4,
  5. Jonathan S. Weissman1,2

Published Online: 1 JUL 2013

DOI: 10.1002/0471142727.mb0418s103

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. and Weissman, J. S. 2013. Genome-Wide Annotation and Quantitation of Translation by Ribosome Profiling. Current Protocols in Molecular Biology. 103:II:4.18:4.18.1–4.18.19.

Author Information

  1. 1

    Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California

  2. 2

    California Institute for Quantitative Biosciences, San Francisco, California

  3. 3

    Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland

  4. 4

    Department of Biology, The Johns Hopkins University, Baltimore, Maryland

Publication History

  1. Published Online: 1 JUL 2013
  2. Published Print: JUL 2013

Abstract

Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. A protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing is presented here. This ribosome-profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes is described. The protocol described requires 5 to 7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis requires an additional 4 to 5 days. Curr. Protoc. Mol. Biol. 103:4.18.1–4.18.19. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • genomics;
  • translation;
  • next-generation;
  • sequencing