Ion Torrent Proton sequencing; RNA-Seq of Brachypodium distachyon callus treated with methyl-jasmonate
RNA-Seq of Brachypodium distachyon callus treated with methyl-jasmonate
Immature Brachypodium embryos were removed from sterilised seeds by dissection, and were transferred to callus induction media (CIM; 4.43 g/l LS salts (Duchefa Biochemie), 30 g/l sucrose (Fisher Scientific), 0.6 mg/l CuSO4, 2.5 mg/ml 2,4-D, 0.2% (w/v) PhytagelTM, pH 5.8). The embryos were incubated at 28 °C in the dark to generate callus, which was subcultured every 2-3 weeks to generate a sufficient quantity. Brachypodium calli were transferred onto plates of CIM containing 50 μM meJA, or a mock control solution. Samples were taken at 24 h, 48 h, 4 d and 8 d. Four biological replicates were harvested per treatment at each timepoint. Each biological replicate consisted of three plates (each containing 36 calli) that were pooled. Around 10-50 mg fresh tissue was suspended in 1 ml TRI Reagent® and then centrifuged (12,000 g, 4 °C, 10 min). The supernatant was added to 200 μl chloroform, and incubated at room temperature for 2-3 min. The samples were centrifuged (10,000 g, 4 °C, 15 min). To the upper phase, ½ volume of isopropanol, and an equal volume of 0.8 M aqueous sodium citrate, 1.2 M aqueous NaCl was added. The samples were incubated at room temperature for 10 min, and centrifuged (10,000 g, 4 °C, 10 min). The pelleted RNA was washed with 1 ml 75% (v/v) aqueous ethanol (vortexed, centrifuged 10,000 g, 4 °C, 10 min), and evaporated to dryness under vacuum. RNA was resuspended in nuclease free water. RNA concentration was determined on a NanoDrop 1000 Spectrophotometer (Thermo Scientific). RNA-seq libraries were constructed the Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific) according to the supplied protocol.
Experimental Factor: mock control
treatment
Experimental Factor: 2
time