Format

Send to

Choose Destination
Methods. 2017 Aug 15;126:112-129. doi: 10.1016/j.ymeth.2017.05.028. Epub 2017 Jun 1.

Transcriptome-wide measurement of translation by ribosome profiling.

Author information

1
Department of Molecular and Cell Biology, Center for RNA Systems Biology, California Institute for Quantitative Biosciences, University of California, Berkeley, 16 Barker Hall # 3202, Berkeley, CA 94720-3202, USA. Electronic address: mcglincy@berkeley.edu.
2
Department of Molecular and Cell Biology, Center for RNA Systems Biology, California Institute for Quantitative Biosciences, University of California, Berkeley, 16 Barker Hall # 3202, Berkeley, CA 94720-3202, USA. Electronic address: ingolia@berkeley.edu.

Abstract

Translation is one of the fundamental processes of life. It comprises the assembly of polypeptides whose amino acid sequence corresponds to the codon sequence of an mRNA's ORF. Translation is performed by the ribosome; therefore, in order to understand translation and its regulation we must be able to determine the numbers and locations of ribosomes on mRNAs in vivo. Furthermore, we must be able to examine their redistribution in different physiological contexts and in response to experimental manipulations. The ribosome profiling method provides us with an opportunity to learn these locations, by sequencing a cDNA library derived from the short fragments of mRNA covered by the ribosome. Since its original description, the ribosome profiling method has undergone continuing development; in this article we describe the method's current state. Important improvements include: the incorporation of sample barcodes to enable library multiplexing, the incorporation of unique molecular identifiers to enable to removal of duplicated sequences, and the replacement of a gel-purification step with the enzymatic degradation of unligated linker.

KEYWORDS:

High-throughput sequencing; RNA; RNA-sequencing; Ribosome; Ribosome profiling; Translation

PMID:
28579404
PMCID:
PMC5582988
DOI:
10.1016/j.ymeth.2017.05.028
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center