title: NextSeq 500 sequencing; Distinct Cellular Strategies Determine Sensitivity to Mild Drought of Arabidopsis Natural Accessions
design: Distinct Cellular Strategies Determine Sensitivity to Mild Drought of Arabidopsis Natural Accessions
library_construction_protocol: Eighty seedlings (distributed over nine to ten pots) were grown per ecotype per treatment per replicate. At 11 DAS, seedlings were harvested at 6 PM and stored in RNA-later (Ambion) solution. The third leaf was harvested by micro-dissecting, then pooled and flash frozen in liquid nitrogen. In total, three biological replicates were performed. the third leaf was harvested by micro-dissecting, then pooled and flash frozen in liquid nitrogen. For the experiment of MD treatment in soil, seeds were sown in pots, which were filled up to 85 g ± 0.5 g of Saniflor soil (Van Isreal N.V., Geraardsbergen, Belgium) with an absolute water content of on average 70%. After 5 nights of stratification at 4°C in darkness, the pots were moved to a growth chamber (21°C and 16-h day/8-h night cycles). At 4 DAS, the pots were transferred to the automated phenotyping platform WIWAM (21°C and 16-h day/8-h night cycles) and randomized to homogenously mix the natural accessions and treatments. The controlled MD treatment was initiated at 6 DAS. Control-treatment pots were kept at 2.2 gwater/gsoil. The MD-treated pots initially contained 2.2 gwater/gsoil and their relative water content was further decreased to 1.2 gwater/gsoil, which was reached at 11 DAS. All plants were watered daily from 4 days after stratification (DAS) until 5 DAS with a WW regime of 2.2 gwater/gsoil. At 6 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 11 DAS. RNA extraction was done using Trizol (Invitrogen) combined with the RNeasy Mini Kit (Qiagen), following the manufacturer's instructions. In total, three biological replicates were performed. Sequence-libraries of each sample were equimolarly pooled and sequenced on Illumina NextSeq 500 instrument (v2.5, High Output, 75 bp, Single Reads: 76-8-8-0) at the VIB Nucleomics core (www.nucleomics.be).