Simple and inexpensive ribosome profiling analysis of mRNA translation.

Published

Journal Article

The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling.

Full Text

Duke Authors

Cited Authors

  • Reid, DW; Shenolikar, S; Nicchitta, CV

Published Date

  • December 2015

Published In

Volume / Issue

  • 91 /

Start / End Page

  • 69 - 74

PubMed ID

  • 26164698

Pubmed Central ID

  • 26164698

Electronic International Standard Serial Number (EISSN)

  • 1095-9130

International Standard Serial Number (ISSN)

  • 1046-2023

Digital Object Identifier (DOI)

  • 10.1016/j.ymeth.2015.07.003

Language

  • eng