Illumina HiSeq 2500 sequencing; RNA-Seq of developing shoot apical meristems of barley cultivar Golden Promise (null segregant and Ubi::HvFT3 overexpressor) grown under long- and short-day photoperiods
RNA-Seq of developing shoot apical meristems of barley cultivar Golden Promise (null segregant and Ubi::HvFT3 overexpressor) grown under long- and short-day photoperiods
RNA was isolated from tissue of the main shoot apices harvested from transgenic barley Ubi::HvFT3 and null segregant plants grown under long (16h/8h, light/dark) and short day (8h/16h, light/dark) conditions. Apex tissue was harvested at the spikelet initiation and lemma primordium stages two hours before the end of the light period. Leaves surrounding the apex were removed manually using a microsurgical stab knife. For each of three biological replicates at least 10 apices were pooled. Wild type Golden Promise, transgenic Ubi::HvFT3 and null segregant plants were grown under long (16h/8h, light/dark) and short day (8h/16h, light/dark) conditions in a controlled plant chamber (20°C day/16°C night) and in soil (Einheitserde). For RNA isolation, the pooled MSA were ground, dissolved in 500 mL TRIzol reagent (Invitrogen), and incubated for 5 min at room temperature. Next, 100 mL of chloroform was added, and the sample was homogenized, incubated for 2 min, and centrifuged for 15 min at 4°C. After phase separation, isopropanol was added to the aqueous phase, which was subsequently incubated for 10 min at room temperature and further purified using an RNA easy Micro Kit (Qiagen). Before RNA sequencing, the residual DNA was removed using a DNA-free kit (Ambion), and the quality of the RNA was tested using a bio analyzer (Agilent). The Illumina cDNA libraries were prepared according to the TruSeq RNA sample preparation (version 2; Illumina).
Experimental Factor: sampling time point
Waddington stage 3.5
Experimental Factor: phenotype
wild type
Experimental Factor: growth condition
short day length regimen