GSM2254600: 8WT4Cork; Populus tremula x Populus alba; RNA-Seq
Total RNA was extracted from 1 g tissue from each. Each sample was mechanically ground in liquid nitrogen until a fine powder was obtained. To this tissue 3 % insoluble polyvinylpyrrolidinone (PVPP) was added and further ground in an extraction buffer containing 2% hexadecyltrimethylammonium bromide (CTAB), 2.0 M sodium chloride, 0.5 g/L spermidine, 100 mM Tris-HCl, 25 mM EDTA and 3% ß-mercaptoethanol pre-warmed to 65 °C. The mixture was then incubated at 65 °C for 10 minutes before extraction with 24:1 chloroform:isoamyl alcohol. The RNA was precipitated by the addition of ¼ volume 10 M LiCl, incubation at 4 °C for 6 hours followed by incubation at -20 °C overnight. The RNA was then harvested by centrifugation (10 000 x g) at 4 °C for one hour, removing aqueous phase, and washing in 70 % ethanol. Pellets were dried, re-dissolved in RNase- free water, and stored at -80 °C until use. Unstranded RNA protocol