GSM8862378: WT Polysomes #2; Drosophila melanogaster; RNA-Seq
Polysome profiling of ovaries was carried out with modifications based on established protocols (44). In brief, we dissected ovaries from approximately 100 YWT flies (UAS-Dcr2; nosGAL4) or around 275 pairs of ovaries from zfrp8 experimental flies using PBS. The dissected ovaries were immediately flash-frozen in liquid nitrogen. The frozen tissue was then homogenized in lysis buffer using a motorized pestle, with 20% of the lysate reserved for mRNA extraction and library preparation as previously described. The remaining lysate was loaded onto sucrose gradients (10–45%) supplemented with cycloheximide, using Beckman Coulter 9 of 16 × 3.5 PA tubes (#331372), and centrifuged at 35,000g in an SW41 rotor for 3 hours at 4°C. After centrifugation, the gradients were fractionated using a density gradient fractionation system. RNA was extracted from the fractions with acid phenol–chloroform, precipitated overnight, and the resulting pellet was resuspended in 20 μl of water, treated with TURBO DNase, and used for library preparation as outlined above