D. miranda MSH22 virgin female ovary RNA-seq
We extracted the total RNAs from whole body or dissected tissues of D. miranda (Qiagen) and prepared the RNA-seq libraries following the standard Illumina protocol. Briefly, we used Dynal oligo(dT) beads (Invitrogen) to isolate poly(A) mRNA from the total RNA samples. We then fragmented the mRNA by using the RNA fragmentation kit from Ambion, followed by first- and second-strand cDNA synthesis using random hexamer primers (Invitrogen). We complemented the cDNA synthesis by an “end repair” reaction using T4 DNA polymerase and Klenow DNA polymerase for 30 min at 20°C. We then added a single A base to the cDNA molecules by using 3'-to-5' exo-nuclease, and ligated the Illumina adapter. The fragment were then subject to size selection on a 2% gel and purification (Qiagen). We finally amplified the cDNA fragments by PCR reaction and examined the libraries by Bioanalyzer (Aglient).