BLANCO Flavio Antonio
capítulos de libros
Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes
REYNOSO, MAURICIO; JUNTAWONG, PIYADA; LANCIA, MARCOS; BLANCO, FLAVIO; BAILEY-SERRES, JULIA; ZANETTI, MARÍA EUGENIA
Methods in Molecular Biology, Plant Functional Genomics
Springer Science Bussiness Media New York
Año: 2014; 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 alternative 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.