RNAseq of temperature and osmotic stress on detached root of Arabidopsis thaliana col-0 and snrk2.2/2.3
RNAseq of temperature and osmotic stress on detached root of Arabidopsis thaliana col-0 and snrk2.2/2.3
Detached roots were harvested in dark conditions using a green safe-light and immediately frozen in liquid nitrogen. Each sample comprised material from 40 roots, which were stored at -80 °C until RNA extraction. seeds were surface-sterilised and then sowed on 1/10th Murashige and Skoog medium including vitamins (Duchefa Biochemie), supplemented with 0.5 g/ l MES (Duchefa Biochemie) and 0.8% Diashin Agar (Duchefa Biochemie), pH5.8. After 3-4 days of stratification in dark at 4˚C, plates with seed were then placed vertically at 90˚ in a growth chamber for 3 days in white light (100µE, 16h photoperiod, 20˚C) to stimulate germination. At day 3, roots were detached and placed on the same media, supplemented with 5 mM sucrose (Duchefa Biochemie) unless otherwise stated. For osmotic stress and ABA treatments, the medium was additionally supplemented with 25 mM sorbitol (Duchefa Biochemie), 25 mM mannitol (Duchefa Biochemie), 12.5 mM NaCl (Duchefa Biochemie) or 30 nM ABA (Duchefa Biochemie) unless otherwise stated. Root tip position was marked after detachment, and roots were grown in darkness for another 4 days at either 20˚C or 28˚C.  seeds were surface-sterilised and then sowed on 1/10th Murashige and Skoog medium including vitamins (Duchefa Biochemie), supplemented with 0.5 g/ l MES (Duchefa Biochemie) and 0.8% Diashin Agar (Duchefa Biochemie), pH5.8. After 3-4 days of stratification in dark at 4˚C, plates with seed were then placed vertically at 90˚ in a growth chamber for 3 days in white light (100µE, 16h photoperiod, 20˚C) to stimulate germination. At day 3, roots were detached and placed on the same media, supplemented with 5 mM sucrose (Duchefa Biochemie) unless otherwise stated. For osmotic stress and ABA treatments, the medium was additionally supplemented with 25 mM sorbitol (Duchefa Biochemie), 25 mM mannitol (Duchefa Biochemie), 12.5 mM NaCl (Duchefa Biochemie) or 30 nM ABA (Duchefa Biochemie) unless otherwise stated. Root tip position was marked after detachment, and roots were grown in darkness for another 4 days at either 20˚C or 28˚C.  Samples were ground in Retsch shaker mill with two 1/8” metal ball bearings per sample, at a frequency of 25 Hz for 30 seconds twice. Total RNA was extracted using RNeasy Plant Mini kit (Qiagen), according to manufacturer instructions. RNA poly-A enrichment library preparation were carried out by Novogene.