title: Illumina HiSeq 2000 sequencing; Time of day determines Arabidopsis transcriptome and growth dynamics under mild drought
design: Time of day determines Arabidopsis transcriptome and growth dynamics under mild drought
library_construction_protocol: Four third leaves were harvested The third true leaf, composed of proliferating and expanding cells at the moment of harvesting were dissected and flash frozen in liquid nitrogen immediately upon harvest. The seeds were sown in 85g +/- 1g of Saniflor compost (Van Isreal N.V., Geraardsbergen, Belgium) with an absolute water content of +/- 70%. The seeds were covered with plastic foil until 5 DAS, when the automated watering was started. When runs with mutants were performed, the pots were randomized to homogenously mix mutant and wild-type plants. All plants were watered daily from 5 days after stratification (DAS) until 11 DAS with a WW regime of 2.2 gwater/gsoil. At 12 DAS, half of the pots (random positions) were maintained at the WW regime, while the other half were not watered until the end of the experiment at 17 DAS. Arabidopsis Col-0 were grown under a long-day regime (light from 6AM until 10PM, intensity 110-120 μmol m-2 s-1) at 21°C. All reported experiments were performed on the WIWAM platform (http://www.wiwam.be). In our setup, four seedlings were grown per pot in order to grow 864 seedlings simultaneously on the platform. All plants were watered daily from 5 days after stratification (DAS) until 11 DAS with a WW regime of 2.2 gwater/gsoil. At 12 DAS, half of the pots (random positions) were maintained at the WW regime, while the other half were not watered until the end of the experiment at 17 DAS. RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s instructions. DNase treatment was performed on-column with the RNase-free DNase kit (Promega). The samples were further purified on column with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Samples were eluted in RNase-free water. Library preparation was done with the TruSeq RNA Sample Preparation Kit (Illumina). Quality was checked with the Bioanalyzer 2100 (Agilent), and clusters were generated through amplification using the TruSeq SE Cluster Kit (Illumina).