Deep sequencing and high-throughput analysis of PIWI-associated small RNAs

•piRNAs can be identified by small RNA-seq analysis of PIWI protein-associated RNAs.•Directional RNA-seq profiles can be overlaid onto small RNA-seq data.•The methodology can be applied to a wide range of species and sample types.

Small RNAs are now known to be major regulatory factors of gene expression. Emerging methods based on deep-sequencing have enabled the analysis of small RNA expression in a high-throughput manner, leading to the identification of large numbers of small RNAs in various species. Moreover, profiling small RNA data together with transcriptome data enables transcriptional and post-transcriptional regulation mediated by small RNAs to be hypothesized. Here, we isolated PIWIL1 (MIWI)-associated small RNAs from mouse testes, and performed small RNA-seq analysis. In addition, directional RNA-seq was performed using Piwil1 mutant mouse testes. Using these data, we describe protocols for analyzing small RNA-seq reads to obtain profiles of small RNAs associated with PIWI proteins. We also present bioinformatic protocols for analyzing RNA-seq reads that aim to annotate expression of piRNA clusters and identify genes regulated by piRNAs.