The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments
By Nicholas T. Ingolia, G. Brar, Silvia Rouskin, Anna M. McGeachy, Jonathan S. Weissman. Nature Protocols, 2012
1,262 citations recorded by the source corpus.
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.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/e2d99c771d6d1b3334e3779505e519776da109b9
How researchers see this paper
The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.
In Molecular Biology
- Jonathan S. Weissman
- Nicholas T. Ingolia
- Gene‐Wei Li
- Benedikt Obermayer
- Carol A. Gross
- D. Burkhardt
- G. Brar
- Matthias Selbach
Citations in the corpus
- Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. (2014, methodology): “The exact length distribution of protected fragments can vary slightly between samples, likely due to differences in digestion conditions (Ingolia et al., 2012).”
- Detecting actively translated open reading frames in ribosome profiling data (2015, methodology): “Custom Unix scripts and BedTools41 were used to create data tracks containing (1) Ribo-seq coverage, (2) P-site positions, (3) RNA-seq coverage and (4) RNA-site positions for different genomic regions.”
- Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. (2014, methodology): “The ribosome-profiling approach involves freezing of cellular translation followed by digestion of all mRNA regions that are not protected by the ribosome (Ingolia et al., 2009, 2012).”