GSM8862344: Control #4; Drosophila melanogaster; RNA-Seq
Ovaries were dissected into PBS and transferred to RNase free microcentrifuge tubes. PBS was removed and 100ul of Trizol was added and ovaries were flash frozen and stored at -80 C. Ovaries were then lysed in the microcentrifuge tube using a plastic disposable pestle. Trizol was added to 1 mL total volume while vigorously shaking the tubes and incubated for 5 min at RT. The samples were centrifuged for 20 min at >13,000 g at 4 C, and the supernatant was transferred to a fresh microcentrifuge tube. 500 ul of chloroform was added, and the samples were vigorously shaken and incubated for 5 minutes at RT. Samples were spun at max speed for 10 minutes at 4 C. The supernatant was transferred to a fresh microcentrifuge tube and ethanol precipitated. Sodium acetate was added, equaling 10% of the volume transferred and 2-2.5 volumes of 100% ethanol were added. The samples were shaken thoroughly and left to precipitate at -20 C overnight. The samples were centrifuged at max speed at 4 C for 15 min to pellet the RNA. The supernatant was discarded and 500 ul of 75% ethanol was added to wash the pellet. The samples were vortexed to dislodge the pellet to ensure thorough washing. The samples were spun at 4 C for 5 min and the supernatant was discarded. The pellets were dried for 10-20 min and then resuspended in 20-50ul of RNAse-free water and the absorbance at 260 was measured on a nanodrop to measure the concentration of each sample. RNA libraries for RNA-seq were prepared using Revvity NEXTFLEX Rapid Directional RNA-seq library prep kit 2.0 following manufacturer's protocol.