Cell identity and function are largely determined by specific gene expression patterns and ultimately by the proteome. Current high-throughput sequencing technologies offer the possibility of quantifying gene expression at high resolution, with minimum input and without the constraints of array-based systems, such as the need for specific probes. In addition, techniques are now available to capture genes that are actively being translated. These techniques use either density gradients or epitope-based immunoprecipitation to purify translating ribosomes and associated mRNAs (i.e., translatomes). More recently, the combination of tissue-specific promoters driving epitope-tagged ribosomes with high-throughput sequencing has allowed the identification of genes and networks unique to specific cell types. Translatome analyses have the potential to unravel genetic programs and cellular responses to environmental stresses at cell-specific resolution. This unit describes steps for the use of epitope-based immunoprecipitation to purify translating ribosomes from soybean and the recovery of mRNA for downstream applications such as gene expression analysis. © 2016 by John Wiley & Sons, Inc.
Keywords: Ribo-Seq; TRAP; monosomes; polysomes; ribosome immunoprecipitation; translatome.
Copyright © 2016 John Wiley & Sons, Inc.