Plants were flash-frozen in liquid nitrogen immediately upon harvest. RNAlater®-ICE (Ambion®), which prevents RNA from degrading, was cooled at -70°C, added to the samples and was allowed to penetrate the tissue at -20°C for 5 days. The third leaf was collected by microdissection under a microscope. Samples were microdissected in a Petri dish on dry ice to keep the samples below room temperature. Arabidopsis thaliana (L.) Heynh accessions were grown in a growth chamber under controlled environmental conditions (21°C, 55% relative humidity, 16¬ h day/ 8 h night, 110-120 μmol m-2 s-1 light intensity). Plants were germinated in soil-filled 96-well plates after four days of stratification at 4°C in the dark. Seedlings were transferred to pots at four days after stratification (DAS). Average-sized seedlings were selected using an in-house developed image algorithm in order to reduce seedling size effects at the beginning of the experiment. Before transfer, the relative water content of the pots was set at 1.2 g H2O/g dry soil for the mild drought treatment, the control condition was set at 2.2 g H2O/g dry soil. Once the seedlings were transferred to pots, they were placed on the automated phenotyping platform WIWAM (Skirycz et al., 2011b). The water content of the soil was kept constant until 10 DAS, after which it was lowered daily to target 0.7 g H2O/g dry soil for the mild drought treated plants (Fig. 1D). Pictures of the rosette of each plant were taken daily until 21 DAS and analyzed for the projected rosette area (PRA) (Skirycz et al., 2011b). Dissected leaves were ground with a Retsch machine (Retsch) and 3-mm metal balls. Samples were obtained from 3 independent biological repeats. RNA was extracted with TriZol (Invitrogen) according to the manufacturer’s instructions. RNA samples were subjected to DNA digestion with RNase-free DNase I kit (Qiagen), and subsequently, impurities were removed with the RNeasy mini kit (Qiagen). Library preparation was done using the TruSeq RNA Sample Preparation Kit v2 (Illumina). In brief, polyA containing mRNA molecules are reverse transcribed, double-stranded cDNA is generated and adapters are ligated. After quality control using 2100 Bioanalyzer (Agilent), clusters are generated through amplification using the TruSeq PE Cluster Kit v3-cBot-HS kit (Illumina) followed by sequencing on a Illumina HiSeq2000 with the TruSeq SBS Kit v3-HS (Illumina).
Experimental Factor: Jm-0
ecotype
Experimental Factor: control
environmental stress
Experimental Factor: R1
block