Transcriptomics

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299

The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments


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. Additionally, 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 pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library. Ribosome profiling in cultured mammalian cells under three different footprinting conditions

ORGANISM(S): Homo sapiens  

SUBMITTER: Nicholas T Ingolia   Jonathan S Weissman  Anna M McGeachy  Silvi Rouskin  Gloria A Brar 

PROVIDER: E-GEOD-37744 | ArrayExpress | 2012-07-26

SECONDARY ACCESSION(S): GSE37744SRP012648PRJNA163111

REPOSITORIES: GEO, ArrayExpress, ENA

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Publications

The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.

Ingolia Nicholas T NT   Brar Gloria A GA   Rouskin Silvia S   McGeachy Anna M AM   Weissman Jonathan S JS  

Nature protocols 20120726 8


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 usin  ...[more]

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