Whitworth GB and Watson FL (2023), Translating Ribosome Affinity Purification (TRA...

XB-ART-59578

Methods Mol Biol 2023 Jan 01;2636:279-310. doi: 10.1007/978-1-0716-3012-9_16.

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

Whitworth GB , Watson FL .

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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.

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Species referenced: Xenopus tropicalis Xenopus laevis
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Fig. 1 Stepwise diagram showing details of steps required for preparing the affinity matrix. Preparing the affinity matrix (AM) and control affinity matrix (AM-ct) requires sequentially binding the three affinity matrix components [Streptavidin MyOne T1 magnetic beads (beads), the biotinylated protein L (BP-L), and 100 μg of Htz anti-GFP antibody (mAb) lots (50 μg of 19C8 and 50 μg of 19F7)] in three distinct steps (1–3). The relative ratio of the affinity matrix components needs to be optimized for distinct TRAP-extracted tissues during preliminary pilot experiments (see Notes 18, 19 and 20). The overall volume of liquid to beads should be the same as the volume in which beads arrive from manufacturer (see asterisk and Note 24). Handle beads gently; all resuspension steps are performed by slowly and gently pipetting up and down four times using a P1000 (do not introduce air bubbles; do not vortex) (1e; 2e; 3d; 3j). Following binding of anti-GFP to the biotinylated protein L-coated beads (3g), wash the AM (beads–BP-L–mAB) and AM-ct (beads–BP-L) three times with low-salt buffer (L-S buffer; 3h). After the third and final L-S buffer wash (3h, i), divide the affinity matrix into pre-labeled tubes corresponding the total number of IP samples, and place individual samples on magnet (3i). Discard supernatant and resuspend each sample containing either the AM or AM-ct in final 200 μL volume of L-S buffer (3j). Place samples on ice until sample lysates are ready to be immunoprecipitated (3k). Beads can be prepared up to 2 weeks prior to use (see Note 22)

Fig. 2 Schematic diagram of the major stages for data analysis. File formats typically used at each step are shown above the arrows, possibly optional steps shown in green, and software discussed here are listed at the bottom of the figure

References [+] :

Ashburner, Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. 2000, Pubmed