title: RNA-seq of Drosophila melanogaster 2-4 days old adult female ovaries
design: Total RNA was extracted from 2-4 days old adult female ovaries (after pupae eclosion), whose female germ line was specifically depleted for salsa (salsa RNAi) or negative control (mCherry RNAi). Extraction was performed following standard procedures (PureLink RNA Mini Kit, Ambion, NY, USA). Genomic DNA was removed from RNA samples using PureLink DNase (Invitrogen) on-column method. Experion Electrophoresis (Bio-Rad, California, USA) was used to assess the integrity and quantity of isolated RNA.After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. First, the mRNA is fragmented randomly to short fragments of 250-300 bp in fragmentation buffer, then cDNA is synthesized by using mRNA template and random hexamers primer. After the first strand was synthesized, a custom second-strand synthesis buffer (Illumina), dNTPs (dUTP, dATP, dGTP and dCTP) and DNA polymerase I were added to initiate the second-strand synthesis. Then followed by purification by AMPure XP beads, terminal repair, polyadenylation, sequencing adapter ligation, size selection and degradation of second-strand U-contained cDNA by the USER enzyme. The strand-specific cDNA library was generated after the final PCR enrichment.