SmallRNA-seq analysis of Xenopus germline

Ovaries from adult female frogs were cut into small pieces, flash-frozen in liquid nitrogen, and then mushed in a frozen. Testes from adult male frogs were flash-frozen in liquid nitrogen. Small RNAs were purified using small RNA isolation kit (ISOGEN II, Nippon gene, Japan) by the manufacturer's protocol except for the percentage change of ethanol form 75 to 90. piRNAs were separated on a 15% polyacrylamide TBE-Urea gel, and 20 - 50 bases were excised from the gel. Then they were recovered by small-RNA PAGE recovery kit (Zymo research, California, USA). Sequencing library was prepared using Illumina v1.9 library protocol. Adapter sequences were trimmed using trim-galore, trimmomatic (SE SLIDINGWINDOW:1:10 MINLEN:20 SLIDINGWINDOW:10:20 MINLEN:20, SE LEADING:20 TRAILING:20 SLIDINGWINDOW:10:20 MINLEN:20) and cutadapt (-b "A{10}" -b "G{10}" -b "C{10}" -b "T{10}").