Illumina HiSeq 4000 paired end sequencing; RNA-seq of Norway spruce seedlings exposed to gamma radiation
RNA-seq of Norway spruce seedlings exposed to gamma radiation
Shoots of the seedlings were harvested in liquid nitrogen after 48 h gamma exposure and stored at -80°C until analyses.   Seeds were surface sterilized in 1% sodium hypochlorite for 5 min, rinsed five times in distilled water and placed on a filter paper for drying. All seeds were evenly sown on 1/2 MS medium ((Murashige & Skoog 1962); Duchefa Biochemie, Harleem, Netherland) with 0.8% agar (Plant agar, Sigma-Aldrich) in  petri dishes of 5 cm diameter. The seeds were then germinated for 6 days at 20°C under a photon flux density of 30 μmol m-2 s-1 at 400-700 nm (TL-D 58W/840 lamps, Phillips) in a 16 h photoperiod. Six days old seedlings were exposed to gamma radiation for 48h at gamma dose rates 1, 10, 40 or 100 mGy/h, using the FIGARO low dose gamma irradiation facility (60Co; 1173.2 and 1332.5 keV γ-rays) at Norwegian University of Life Sciences (Hansen et al. Submitted; Lind et al. 2018; Lind et al. In press). During the gamma irradiation, the room temperature was set at 20˚C±1°C with a 12 h photoperiod with a photon flux density of 55 μmol m-2 s-1 provided by high pressure metal halide lamps (HPI-T Plus 250W lamps, Phillips). Control samples not exposed to gamma radiation were positioned outside the radiation sector, shielded by lead walls, in the same room and under same light and temperature conditions. Total RNA was extracted using the Masterpure Plant RNA Purification Kit (Epicenter, Madison, WI, USA) following the manufacturer`s instructions except that 0.5 % polyvinylpyrrolidone (PVP), mw 360 000, Sigma Aldrich, Steinheim, Germany) was added to the extraction buffer and that 3 µl beta-mercaptoethanol per sample replaced the 1,4-dithiothreitol (DTT) in the manufacturer`s protocol. RNA quality was assessed using an Agilent 2100 Bioanalyzer with an RNA 144000 NanoKit (Agilent technologies, Palo Alto, CA, USA) and the quantity of isolated RNA was measured by a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA).   Strand-specific 20xTruseqTM RNA library preparation kit.