Genome-Wide Annotation and Quantitation of Translation by Ribosome Profiling

Genome-Wide Annotation and Quantitation of Translation by Ribosome Profiling

Genome-Wide Annotation and Quantitation of Translation by Ribosome Profiling

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.

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