Translating Ribosome Affi nity Purifi cation (TRAP) Followed by RNA Sequencing Technology (TRAP-SEQ) for Quantitative Assessment of Plant Translatomes

REYNOSO MAURICIO ALBERTO; PIYADA JUNTAWONG; LANCIA MARCOS; BLANCO FLAVIO ANTONIO; BAILEY-SERRES JULIA; ZANETTI MARÍA EUGENIA

METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.)

Springer

Año: 2015 p. 185 - 207

1064-3745

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 affi nity purifi cation 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.