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

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. PIWIL1-associated small RNA-seq from mouse testes (two replicates), and directional RNA-seq from Piwil1 mutant mouse (Piwil1+/- and Piwil1-/-).现已证实，小RNA（small RNAs）是基因表达的核心调控因子。基于深度测序（deep-sequencing）的新兴技术已实现小RNA表达的高通量分析，助力在多种物种中鉴定出大量小RNA。此外，联合分析小RNA表达谱数据与转录组数据，可推断小RNA所介导的转录及转录后调控机制。本研究从小鼠睾丸中分离得到PIWIL1（MIWI）结合的小RNA，并开展小RNA测序（small RNA-seq）分析；同时以Piwil1突变型小鼠的睾丸为样本，完成定向RNA测序（directional RNA-seq）分析。基于上述实验数据，我们详述了用于分析小RNA测序读段、以获取PIWI蛋白结合小RNA表达谱的实验方案；同时提供了生物信息学分析流程，用于解析RNA测序读段，以注释piRNA（Piwi-interacting RNA）簇的表达水平，并鉴定受piRNA调控的靶基因。本数据集包含：小鼠睾丸的PIWIL1结合小RNA测序数据（含2次生物学重复），以及Piwil1突变型小鼠（Piwil1+/-和Piwil1-/-）的定向RNA测序数据。