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.
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