Ingolia et al. 2012 (PRJNA163111)

Title	The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments
Organism
Number of Samples	3
Release Date	2012/07/26 00:00
Sequencing Types
Protocol Details

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Title
Authors	Ingolia NT,Brar GA,Rouskin S,McGeachy AM,Weissman JS
Journal	Nature protocols
Publication Date	2012 Jul 26
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. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. 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. In addition, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pretreating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5-7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis require a further 4-5 days.
PMC	PMC3535016
PMID	22836135
DOI

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