GSM3374902: Young nosGAL4-Poly Trial 1; Drosophila melanogaster; OTHER
~80 ovaries were dissected in PBS supplemented with cycloheximide and frozen immediately with liquid nitrogen. Tissue was homogenized in 200 μl of cold lysis buffer consisting of 1x Polysome buffer supplemented with 1% Triton-X and 1 protease inhibitor pill per 10 ml of buffer. The lysate was centrifuged at 15,000 x g at 4°C for 10 minutes. 20% of lysate was kept aside for “Input RNA” libraries. 750μl of cleared lysate was loaded onto 10-50% sucrose gradients (500 mM KCl; 15 mM Tris-HCl, pH 7.5; 15 mM MgCl2; and 100 μg/ml cycloheximide) in Beckman Coulter 9/16x3.5 PA tubes (Cat. #331372). Gradients were centrifuged at 35,000xg using a SW41 rotor for 3 hours at 4°C. Gradients were fractionated on a Brandel flow cell (Model #621140007) at 0.75 mls/min and 750μl was collected for each fraction with the sensitivity settings at 0.5 Abs. RNA was extracted from the fractions using standard acid phenol: chloroform extraction. The RNA pellet was washed with 80% ethanol and then air-dried. After air-drying the pellet was dissolved in 10 μl of nuclease-free water. RNA samples were subjected to a DNase treatment using the TURBO DNA-free Kit by Life Technologies (AM1907). poly(A)+ RNA was isolated by double selection with poly-dT beads, using the Turbo DNase treated samples, which is then followed by first- and second-strand synthesis. Sequencing libraries were prepared using NEXTflex Rapid Illumina DNA-Seq Library Prep Kit (Bioo Scientific). Samples were single-end sequenced on a NextSeq 500. Bioo Scientific Manufactures protocol
GEO Accession
GSM3374902