Chapter 9: Translating Ribosome Affinity Purification (TRAP) Followed by RNA Sequencing Technology (TRAP-SEQ) for Quantitative Assessment of Plant Translatomes

MAURICIO A. REYNOSO, PIYADA JUNTAWONG, MARCOS LANCIA, FLAVIO A. BLANCO, JULIA BAILEY-SERRES, AND MARÍA EUGENIA ZANETTI

Plant Functional Genomics: Methods and Protocols, Methods in Molecular Biology,

Springer Science+Business Media New York

Lugar: New York; Año: 2015; p. 185 - 207

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.