HITS-CLIP (CLIP-Seq) for mouse Piwi proteins.

Piwi proteins, such as Aubergine in Drosophila and Miwi and Mili in mice, form a major subclade of the Argonaute family, which comprise a distinct class of RNA-binding proteins (RBPs) able to bind small RNAs. Small RNAs can target complementary RNAs. Piwis are essential for the animal germline and bind Piwi-interacting RNAs (piRNAs) to form pi-RiboNucleoProteins (piRNPs). Although many piRNAs target retrotransposons for safeguarding genome integrity of the germ cell, whether piRNAs can target other mRNAs for regulatory purposes is still under investigation. Here we present the technical protocol for "High Throughput Sequencing after in vivo Crosslinking and Immunoprecipitation" (HITS-CLIP, CLIP-Seq), adapted for mouse Piwi proteins Mili and Miwi. We also provide general recommendations for the application of this protocol for different RBPs and also for the bioinformatic analysis of the deep sequencing data.