RNA-Binding Protein Immunoprecipitation and High-Throughput Sequencing

Abstract

The RNA-binding proteome plays a key role in controlling every step in the life of RNA molecules. Through interaction with dedicated sequence motifs, RNA-binding proteins coordinate processing of cohorts of genes. Understanding such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In Arabidopsis thaliana, RNA immunoprecipitation followed by reverse transcription-PCR has been widely used to test the association of candidate targets with RNA-binding proteins. The detection of unknown target transcripts requires methods operating at the level of the entire transcriptome. Here, we describe a protocol for RNA immunoprecipitation coupled to the generation of libraries from the co-purified RNAs for high-throughput sequencing. This allows determining RNAs associated with RNA-binding proteins in planta at a global scale.