Identifying miRNA Targets Using AGO-RIPseq.

microRNAs (miRNA) are small, noncoding RNAs that bind to messenger RNAs (mRNAs) and regulate their activity. They are, therefore, important posttranscriptional regulators. In recent years it has become clear that miRNAs regulate large genetic networks, rather than single genes, and that one gene can be targeted by several miRNAs. To understand the role of miRNAs in cells or tissues, it is therefore important to analyze the targetome of miRNAs. Here, we present a technique called Argonaute-RNA Immunoprecipitation (AGO-RIP) which takes advantages of the fact that miRNAs and their targets are directly bound by the Argonaute protein family. With this approach quantitative, genome-wide analysis of miRNA targets is possible. In this chapter we describe the RIP-methodology and provide advice for RNA sequencing and bioinformatic analyses.