title: Illumina Genome Analyzer IIx paired end sequencing; RNA-Seq of bacteriophage S-PM2d infecting Synechococcus spp. WH7803 at high light and low light treatments
design: RNA-Seq of bacteriophage S-PM2d infecting Synechococcus spp. WH7803 at high light and low light treatments
library_construction_protocol: 5 x 10^9 cells collected by centrifugation ASW medium, 23degC, 15 umol photons m-2 s-1, 170rpm 3 samples were treated with high light (HL) (210 umol photons m-2 s-1). 3 were kept at the growth irradiance (LL) (15 umol photons m-2 s-1) Total RNA was isolated following Logemann et al. (1987). Briefly, 50ml of Synechococcusvsp. WH7803 was centrifuged at 4000 r.p.m. at 4°C in a Rotanta 46R centrifuge (Hettich,Tuttlingen, Germany) and flash frozen in liquid N2. Pellets were thawed in 1.5 ml of Z buffer (8M guanidinium hydrochloride; 50mM β-mercaptoethanol; 20mM EDTA) at room temperature for 30min. 2 volumes of acidified phenol (pH 4.5) was added and heated to 65˚C for 30min, followed by the addition of chloroform:isoamyl alcohol for 15min. The aqueous phase was transferred to separate microcentrifuge tubes. RNA was precipitated by addition of 1 vol. isopropanol followed by incubation overnight at 4˚C. The tubes were spun at 13,000 r.p.m. in a benchtop centrifuge (Biofuge Pico, Heraeus) at 4˚C for 30mins. The pellet was then washed with 70% (v/v) ethanol and further centrifuged for 15min. Pellets were resuspended in 50ml H20. DNA was removed using the TURBO DNA-freeTM kit(Ambion®/Life Technologies, Carlsbad, U.S.A.) following the manufacturer’s instructions. gDNA contamination was tested by PCR. Any samples that yielded a PCR product were further treated using the TURBO DNA-freeTM kit. ScriptSeq v2.0
attributes.tag: Experimental Factor: stimulus
attributes.value: 15 umol photons m-2 s-1