total RNA was extracted using Trizol reagent (Invitrogen) Total RNA of each sample was used to prepare the piRNA sequencing library, which included the following steps:1) 3'-adaptor ligation;2) 5'-adaptor ligation;3) cDNA synthesis;4) PCR amplification;5) Size selection of ~150 bp PCR amplicons(corresponding to ~30 nt piRNAs)  The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 50 cycles on Illumina HiSeq sequencer following the manufacturer's instructions.