Cloning, Sequencing, and Linkage Analysis of piRNAs

Piwi-interacting RNAs (piRNAs) are 25- to 32-nucleotide-long small RNAs that silence transposable elements (transposons) in animal gonads. piRNAs have a large sequence diversity (over one million different sequences per organism) to target a variety of transposon sequences. This is achieved by flexible and distinct biogenesis pathways that are evolutionarily conserved. In this chapter, I describe a detailed method of purifying and cloning piRNAs from freshly dissected tissue samples, such as fruit fly ovaries, for the high-throughput sequencing. I also describe how to computationally process the sequencing data and interrogate the characteristic pattern of piRNA biogenesis, including ping-pong amplification and head-to-tail phasing.