GSM9365452: Wildtype, replicate 2; Drosophila melanogaster; RNA-Seq
Ovaries were dissected in 1x PBS. RNA isolation was performed using Trizol. Samples were then lysed using a plastic pestle and electric pestle motor, and Trizol volume was brought up to 1ml. 200ul of chloroform was then added, and mixed. Samples were then incubated for 5 minutes at room temperature before centrifugation for 20 minutes at 13000g at 2-8C. The supernatant was transferred to a fresh tube, and sodium acetate (10% of collected supernatant volume), 1ul glyco blue, and 100% ethanol (maximum permissible volume) were added before vortexing. Samples were then incubated for 7 minutes before moving to -20C overnight. RNA pellets were generated from the sample by centrifugation for 15 minutes at 13000g at 4C. RNA pellets were washed with 75% ethanol, then eluted in 20-50ul RNAse free water. Integrity of the RNA was determined by running it on a 1% agarose gel and concentration of RNA was assessed using a Nanodrop. RNA samples were treated with Turbo DNase according to kit instructions (Life Technologies TURBO DNAfree Kit #AM1907). mRNA was enriched through poly(A) tail selection, and libraries were prepared using the Revvity NEXTFLEX Rapid Directional RNA-Seq Kit 2.0 (NOVA-5198-02). mRNA library quality was assessed using Qubit, before sequencing on an Illumina NextSeq2000 (single-end 100 base pair reads). RNA libraries for RNA-seq were prepared using Revvity NEXTFLEX Rapid Directional RNA-seq library prep kit 2.0 following manufacturer's protocol.