RNA-seq of Arabidopsis root samples in response to salt, osmotic and control conditions at multiple timepoints with multiple genotypes
RNA-seq of Arabidopsis root samples in response to salt, osmotic and control conditions at multiple timepoints with multiple genotypes
Roots were dissected using a sharp blade and flash frozen in liquid nitrogen Seeds were wet sterilized in 75% EtOH and 25 % bleach for 5 min, washed twice in 70% EtOH and once in 96 % EtOH. After which the seeds were dried in the laminar flow. 1mL 0.1% agar was added to easy sowing. Seeds were sown on plates and stratified at 4 °C for two days. Plants were grown on half strength MS medium including vitamins (Duchefa) containing 0.1% 2-Morpholinoethanesulfonic acid monohydrate (MES) buffer (Duchefa) and 0.8% Plant agar (Duchefa) and pH was adjusted to 5.8 with KOH. Two 1.5x11 cm 50µM mesh strips (Sefar BV were placed horizontally on the agar plate to facilitate the transfer to treatment plates. Using thin strips allowed the roots to have direct contact with the treatment medium. 60 seeds were sown on one agar plate (30 per mesh strip), stratified and grown vertically at an angle of 90° at 22°C and long day photoperiod (16 hours light, 120µM/m2/s).  After 7 days the plants were transferred to fresh treatment agar plates. For the timeseries, wild type (Col-0) seedlings were treated with 125 mM NaCl (Duchefa), 125 mM KCl (Duchefa), 250 mM Sorbitol (Duchefa), 5 µM ABA (Sigma), or ½ MS medium without supplements (mock) and roots were harvested for RNA isolation after 1, 3, 6, 12 and 24 hours by dissecting the roots with a sharp blade. In parallel, mutants (cop1-4, snrk2.2/2.3, moca1) were treated with 125mM NaCl or mock control and harvested at 1 and 3 hours after stress induction. Wild-type seedlings were also placed on a medium supplemented with 5mM CaCl2 with either 125mM NaCl or mock control and harvested after 1 and 3 hours. Approximately 60 roots from 1 plate were pooled in one sample.  Samples were homogenized using a mixer mill MM400 (Retsch), after which total RNA was isolated using RNeasy Kit (Qiagen). Samples were sent to NovoGene for library preparation and Illumina sequencing in 150bp paired-end mode.