Simple and inexpensive ribosome profiling analysis of mRNA translation.
Methods
; 91: 69-74, 2015 Dec.
Article
en En
| MEDLINE
| ID: mdl-26164698
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ribosomas
/
Biosíntesis de Proteínas
/
ARN Mensajero
/
Análisis de Secuencia de ARN
/
Nucleasa Microcócica
Tipo de estudio:
Guideline
Límite:
Humans
Idioma:
En
Revista:
Methods
Asunto de la revista:
BIOQUIMICA
Año:
2015
Tipo del documento:
Article