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.