title: GSM5368492: rls6_2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Total plant RNA was extracted from 25-d-old plants grown under LD condition using the Trizol reagent (Invitrogen, Carlsbad, USA) and dissolved in RNase-free H2O. To make siRNA library, 30 μg RNA was mixed with equal volume of 2× RPA loading buffer (10 ml of 98% formamide, 200 µl 2 mM EDTA, 10 mg bromophenol blue, 10 mg xylene cyanol) and denatured at 94℃ for 3 min. RNA samples were separated on 15% TBE-PAGE by running at 120 v for 2 h at 4℃, the gel was then stained with 5 µl of Syber Gold (Invitrogen, S11494) for 5 min in 50 ml of 1× TBE buffer (89 mM Tris base, 89 mM boric acid, 1 mM EDTA, pH 8.0). The gel band corresponding to 17-30 nt sRNA was cut and ground in a low-bind 1.5-ml tube with a RNase-free pestle in 500 μl of 0.3 M NaCl. Then the tube was incubated at -80℃ for 30 min at 4℃ overnight with slow rotation.  The gel slurry was filtered through a Spin-X filter (Corning, Cat. 8162) by centrifugation at 20,000 ×g for 1 min at 4℃. The flow through was transferred to two 1.5-ml tubes, each contain 250 μl flow through and was added with  625 µl ethanol, 25 µl NaAc (3 M, pH=5), 1 µl glycogen (Invitrogen, Cat. 10814010). After keeping at -80℃ for 4 h, sRNA was pelleted by centrifugation at 20,000 ×g for 30 min at 4℃. The pellets was washed with 80% ethanol for two times and dissolved in 7 µl of RNase-free H2O. NEBNext® Small RNA library prep kit (E7300)