Illumina HiSeq 1500 sequencing; Establishment of photosynthesis through chloroplast development is controlled by two distinct regulatory phases
Establishment of photosynthesis through chloroplast development is controlled by two distinct regulatory phases
Cells were collected from the culture by filtration, immediately frozen in liquid nitrogen and ground in a mortar. Arabidopsis thaliana (Col-0) cell lines were grown in Murashige and Skoog (MS) medium supplied with 3% (w/v) sucrose, pH 5.7 in the dark at 25°C, shaken at 140 rpm (Pesquet et al., 2010). Cells were subcultured weekly by a 1:10 dilution. For all experiments, 7-day-old cells from dark conditions were subcultured in a 1:10 ratio in MS medium 1% (w/v) sucrose, equilibrated and placed in a growth cabinet under continuous light (150 μmol photons m-2 s-1) and constant rotary agitation. After seven days in the light, cells were pelleted without centrifugation and the medium was replaced. Cells were placed back in the light until 14 days for the final sample. Total RNA was isolated using the mirVana miRNA Isolation kit (Ambion) according to manufacturer ́s instructions. RNA was quantified with a Nanodrop ND-100 spectrophotometer and RNA quality was checked by electrophoresis on a 1% agarose gel. RNA samples were ethanol precipitated and diluted to 500 ng/μL. RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies) and the RNA was quantified with a Qubit 2.0 fluorometer (Thermo-Fisher Scientific), both according to manufacturer's instructions. RNA-Seq libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina), according to manufacturer's instructions, using half-reactions and 200 ng RNA. Successful rRNA depletion was verified by electrophoresis using the High Sensitivity RNA ScreenTape System on the Agilent 2200 TapeStation (Agilent Technologies), according to manufacturer's instructions. Enrichment of cDNA fragments was performed using 11 cycles generating cDNA fragments of ~270 bp.
Experimental Factor: time
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