UNIT 4.18 Genome-Wide Annotation and Quantitation of Translation by Ribosome Profiling
Published Online: 1 JUL 2013
Copyright © 2013 by John Wiley & Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 JUL 2013
- Published Print: JUL 2013
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.