RNA-Seq of D. miranda: spermatneca
We extracted total RNA from dissected tissues (Qiagen) and prepared 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 20ĄC. We then added a single A base to the cDNA molecules by using 3'-to-5' exo-nuclease and ligated the Illumina adapter. The fragments were subjected 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).Ę