GSM9472287: Body, exposed to pathogen and caffeinated ant food, 02; Formica fusca; RNA-Seq
RNA was isolated from each sample with a phenol-chlorophorm extraction method. First, samples were homogenized with 500 µl TRI Reagent (T9424, Sigma-Aldrich, Missouri, United States) with a TissueLyser II (Qiagen, Venlo, Netherlands) for 1min at 30/s. Then, the volume was adjusted to 1 ml with TRI Reagent and the samples were vortexed. A volume of 150 µl of chloroform (C2432, Sigma-Aldrich, Missouri, United States) was added and the samples were vortexed vigorously. The samples were incubated at room temperature for 2 min and then on ice for 5 min. The samples are then centrifuged for 10 min at 14,000 rpm and at 4 °C. The supernatant was then carefully transferred to a new clean tube and 500 µl of isopropanol (190764, Sigma-Aldrich, Missouri, United States) was added. The samples are then vortexed vigorously and stored at -20 °C for approx. 24 h. The samples were centrifuged for 30 min at 14,000 rpm and at 4 °C. The isopropanol was removed and 500 µl of 80 % ethanol was added. The samples were centrifuged for 10 min at 14,000 rpm and at 4 °C. The ethanol was removed, and the samples were spined down to allow a second removal of the remaining solution. The RNA pellets were allowed to air dry for approx. 10 min with open cap. Finally, 40 µl of miliQ H2O were added and the RNA concentration and purity were measured with the Nanodrop Onec (Thermo Fisher Scientific, Massachusetts, United States). RNA extracts that were under 1.8 from the 260/280 ratio were subjected to a clean-up with GeneJET RNA Cleanup and Concentration Micro Kit (K0842, Thermo Fisher Scientific, Massachusetts, United States). Twenty microliters of RNA extracts were used and adjusted with nuclease-free water to reach a volume of 200 µl. Then, the manufacturer protocol was followed. As the amount of RNA was large, 20 µl of nuclease-free water was added instead of 10 for the final elution volume. RNA concentration and purity were measured with a Nanodrop and the RNA extracts with low purity levels were subjected to another round of clean-up. Library construction was performed by Novogen using Illumina NovaSeq™ X Plus instruments and a paired-end 150 bp sequencing strategy.