Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes

Methods Mol Biol. 2015;1284:185-207. doi: 10.1007/978-1-4939-2444-8_9.

Abstract

Translating Ribosome Affinity Purification (TRAP) is a technology to isolate the population of mRNAs associated with at least one 80S ribosome, referred as the translatome. TRAP is based on the expression of an epitope-tagged version of a ribosomal protein and the affinity purification of ribosomes and associated mRNAs using antibodies conjugated to agarose beads. Quantitative assessment of the translatome is achieved by direct RNA sequencing (RNA-SEQ), which provides accurate quantitation of ribosome-associated mRNAs and reveals alternatively spliced isoforms. Here we present a detailed procedure for TRAP, as well as a guide for preparation of RNA-SEQ libraries (TRAP-SEQ) and a primary data analysis. This methodology enables the study of translational dynamic by assessing rapid changes in translatomes, at organ or cell-type level, during development or in response to endogenous or exogenous stimuli.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Fractionation / methods
  • Gene Library
  • High-Throughput Nucleotide Sequencing* / methods
  • Plants / genetics*
  • Plants / metabolism*
  • Plants, Genetically Modified
  • Polyribosomes / metabolism
  • Protein Biosynthesis*
  • RNA, Messenger / genetics*
  • RNA, Messenger / isolation & purification
  • RNA, Messenger / metabolism*
  • Ribosomes / metabolism*

Substances

  • RNA, Messenger