Isolation and Library Preparation of Planarian piRNAs.

In planarian flatworms, piRNAs and SMEDWI (Schmidtea mediterranea PIWI) proteins are both essential for the animals' impressive regenerative ability and for their survival. A knockdown of SMEDWI proteins disrupts the specification of the planarian germline and impairs stem cell differentiation, resulting in lethal phenotypes. As the molecular targets of PIWI proteins and thus their biological function are determined by PIWI-bound small RNAs, termed piRNAs (for PIWI-interacting RNAs), it is imperative to study the wealth of PIWI-bound piRNAs using next-generation sequencing-based techniques. Prior to sequencing, piRNAs bound to individual SMEDWI proteins must be isolated. To that end, we established an immunoprecipitation protocol that can be applied to all planarian SMEDWI proteins. Co-immunoprecipitated piRNAs are visualized by using qualitative radioactive 5'-end labeling, which detects even trace amounts of small RNAs. Next, isolated piRNAs are subjected to a library preparation protocol that has been optimized for the efficient capture of piRNAs, whose 3'-ends carry a 2'-O-methyl modification. Successfully prepared piRNA libraries are subjected to Illumina-based next-generation sequencing. Obtained data are analyzed as presented in the accompanying manuscript.

Genome-Wide Analysis of Planarian piRNAs.

In planarian flatworms, the piRNA pathway is operated by three PIWI proteins, termed SMEDWI-1, SMEDWI-2, and SMEDWI-3 (SMEDWI = Schmidtea mediterranea PIWI). The interplay between these three PIWI proteins and their associated small noncoding RNAs, termed piRNAs, fuels the outstanding regenerative abilities of planarians, enables tissue homeostasis, and, ultimately, ensures animal survival. As the molecular targets of PIWI proteins are determined by the sequences of their co-bound piRNAs, it is imperative to identify these sequences by next-generation sequencing applications. Following sequencing, the genomic targets and the regulatory potential of the isolated piRNA populations need to be uncovered. To that end, here we present a bioinformatics analysis pipeline for processing and systematic characterization of planarian piRNAs. The pipeline includes steps for the removal of PCR duplicates based on unique molecular identifier (UMI) sequences, and it accounts for piRNA multimapping to different loci in the genome. Importantly, our protocol also includes a fully automated pipeline that is freely available at GitHub. Together with the piRNA isolation and library preparation protocol (see accompanying chapter), the presented computational pipeline enables researchers to explore the functional role of the piRNA pathway in flatworm biology.