GSM8554602: FCD_3; Ovis aries; RNA-Seq
adipose tissue was thawed in Trizol and homogenized using 3 pulses, 4-6 seconds each. Then, 0.2 mL of chloroform was added per 1 mL of Trizol (0.1 mL per sample). Samples were centrifuged for 15 minutes at 14,000 x g (4 ˚C) and the mixture separated into a lower red phenol-chloroform, interphase, and a colorless upper aqueous solution. The aqueous phase containing RNA was transferred to a new tube very carefully to ensure proteins and other cell components did not contaminate the RNA. Then, 250 µl of isopropanol was added to the aqueous phase (0.5 ml/ ml of Trizol) and incubated for 10 minutes. The sample was centrifuged for 10 minutes at 14,000 x g (4˚C) and the RNA precipitate formed a white gel-like pellet at the bottom of the tube. The supernatant was discarded and the sample was vortexed briefly before centrifugation for 5 minutes at 8000 x g (4˚C). The supernatant was discarded and the RNA pellet was air-dried for 10 minutes. Pellets were resuspended in 50 µl of RNase-free water. The RNA was further purified by Zymo Purelink RNA columns. library was constructed from samples using a Swift RNA Library Kit – (Catalog # R1024) and Swift Dual Combinatorial Indexing Kit – (Catalog # X8096)