title: Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes.
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.
authors.affiliation: Instituto de Biotecnología y Biología Molecular (BBM), FCE-UNLP CCT-CONICET, Calle 115 entre 49 y 50, La Plata, Argentina.
authors.forename: Mauricio A
authors.initials: MA
authors.lastname: Reynoso
authors.forename: Piyada
authors.initials: P
authors.lastname: Juntawong
authors.forename: Marcos
authors.initials: M
authors.lastname: Lancia
authors.forename: Flavio A
authors.initials: FA
authors.lastname: Blanco
authors.forename: Julia
authors.initials: J
authors.lastname: Bailey-Serres
authors.forename: María Eugenia
authors.initials: ME
authors.lastname: Zanetti
mesh_terms: D002458:Cell Fractionation / Q000379:methods; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D010944:Plants / Q000235:genetics* / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger