title: GSM6671048: Ad libitum female brain biol rep 2; Nothobranchius furzeri; RNA-Seq
library_construction_protocol: We isolated RNA from liver and brain using Qiagen RNeasy Mini kit (Qiagen, #74106) according the manufacturer's instructions. The processing order of each organ was randomized. The tissues were transferred to the 1.2 mL Collection Microtubes (Qiagen, #19560) on dry ice in a 4°C cold room to reduce tissue thawing. Next, an autoclaved metal bead (Qiagen, #69997) and 700 µL of QIAzol (Qiagen, #79306) were added to each tube. Two rounds of tissue homogenization were performed on the TissueLyserII machine (Qiagen, #85300) at 25 Hz, 5 min each, at room temperature. The lysate was transferred to new 1.5 mL tubes, 140 µL chloroform (Fisher Scientific, #C298-500) was added, and the tubes were vortexed for 15 sec. After incubation at room temperature for 2-3 min, lysates were centrifuged at 12,000 x g at 4°C for 15 min. The aqueous phase was mixed with 350 µL ethanol (200 Proof, Gold Shield Distributors, #412804) by inverting the tubes 10 times. The mixture was transferred to the RNeasy column, washed with 350 µL RW1 buffer (provided by the RNeasy Mini kit), and treated with DNase I (following the kit's protocol) at room temperature, for 15 min. The column was washed 2 times with 500 µL RPE buffers, and the RNA was eluted with 50 µL nuclease-free water (Invitrogen, #10977023). RNA quality and concentration were measured using an Agilent 2100 Bioanalyzer and the Agilent Nano Eukaryotic RNA Kit (Agilent, #5067-1511). 10 ng of RNA of each sample was used for cDNA synthesis. cDNA synthesis was performed using the Takara SMART-seq v4 PLUS kit (Takara, #634889) according to the manufacturer's instructions. The cDNA libraries were prepared using the Illumina NexteraXT DNA library prep kit (Illumina, #FC-131-1096) based on the manufacturer's protocol. The Illumina Nextera XT Index Kit v2 (Illumina, #131-2001) was used for library indexing.