Translating Ribosome Affinity Purification (TRAP) and Bioinformatic RNA-Seq Analysis in Post-metamorphic Xenopus...

Recent technical advances provide the ability to isolate and purify mRNAs from genetically distinct cell types so as to provide a broader view of gene expression as they relate to gene networks. These tools allow the genome of organisms undergoing different developmental or diseased states and environmental or behavioral conditions to be compared. Translating ribosome affinity purification (TRAP), a method using transgenic animals expressing a ribosomal affinity tag (ribotag) that targets ribosome-bound mRNAs, allows for the rapid isolation of genetically distinct populations of cells. In this chapter, we provide stepwise methods for carrying out an updated protocol for using the TRAP method in the South African clawed frog Xenopus laevis. A discussion of the experimental design and necessary controls and their rationale, along with a description of the bioinformatic steps involved in analyzing the Xenopus laevis translatome using TRAP and RNA-Seq, is also provided.