Small RNA Library Generation Protocol

Small RNA libraries were generated as described previously with slight modifications (McGinn and Czech 2014 (link)). Briefly, 18- to 29-nt-long small RNAs were purified by PAGE from 15 µg of total RNA from ovaries or OSCs. Next, the 3′ adapter (containing four random nucleotides at the 5′ end) (Jayaprakash et al. 2011 (link)) was ligated using T4 RNA ligase 2 and truncated KQ (New England Biolabs). Following recovery of the products by PAGE purification, the 5′ adapter (containing four random nucleotides at the 3′ end) was ligated to the small RNAs using T4 RNA ligase (Ambion). Small RNAs containing both adapters were recovered by PAGE purification, reverse-transcribed, and PCR-amplified. Libraries were sequenced on an Illumina HiSeq 4000. All adapter sequences are in Supplemental Table S6.