GSM7336849: Total RNA-seq of old brain replicate 2; Nothobranchius furzeri; RNA-Seq
Individual brains from the fish were collected and snap-frozen in liquid nitrogen. The protein amount was estimated based on fresh tissue weight (assuming 5% of protein w/w) and ice-cold 1x PBS with protease/ phosphatase inhibitors (Roche,11836170001, 4906837001) was added accordingly to a final concentration of 2 μg/μL. Samples were then vortexed (5 times) before sonication (Bioruptor Plus) for 10 cycles (60 sec ON/30 sec OFF) at the high setting, at 4 °C. The samples were then centrifuged at 3000 xg for 5 min at 4 °C, and the supernatant was transferred to 2 mL Eppendorf tubes. 1.5 mL of ice-cold Qiazol (Qiagen, 79306) reagent was added to 150 μL of homogenate, vortexed five times, and snap-frozen in liquid nitrogen. On the day of the experiment, samples were thawed on ice, vortexed five times, and incubated at room temperature for 5 min before the addition of 300 μL of chloroform. Samples were mixed vigorously, incubated for 3 min at room temperature, and centrifuged at 12000 xg for 20 min at 4 °C. The upper aqueous phase (600 μL) was carefully transferred into a fresh tube and the remaining volume (phenol/chloroform phase) was kept on ice for DNA isolation. The aqueous phase was mixed with 1.1 volume of isopropyl alcohol, 0.16 volumes of sodium acetate (2 M; pH 4.0), and 1 μL of GlycoBlue (Invitrogen, AM9515) to precipitate RNA. After 10 min incubation at room temperature, samples were centrifuged at 12000 xg for 30 min at 4 °C. The supernatant was completely removed, and RNA pellets were washed by adding 80% (v/v) ethanol and centrifuging at 7500 xg for 5 min at 4 °C. The washing steps were performed twice. The resulting pellets were air-dried for no more than 5 min and dissolved in 10 μL nuclease-free water. To ensure full dissolution of RNA in water, samples were then incubated at 65 °C for 5 min, before storage at -80 °C. Total RNA library preparation was done by introducing 500 ng total RNA into Illumina's NEBNext Ultra II directional mRNA (UMI) kit (NEB, E7760S), following the manufacturer's instructions. The quality and quantity of all libraries were checked using Agilent's Bioanalyzer 2100 and DNA 7500 kit (Agilent Technologies, 5067-1506).