Illumina HiSeq 2500 sequencing; RNA-seq of Arabidopsis root samples in response to salt and control conditions
RNA-seq of Arabidopsis root samples in response to salt and control conditions
Roots were dissected using a sharp blade. For seedling pregrowth in petri dishes for all the root phenotyping experiments, Arabidopsis seeds were surface sterilized in 70% (v/v) ethanol for 1 min, and then immersed in 30% (v/v) bleach with triton X-100 (10 µL/50 mL) for 10 minutes and followed by washing with sterilized MQ water for at least six times. Sterilized seeds were sown on 0.5x Murashige and Skoog (MS) (including B5 vitamins, Duchefa Biochemie B.V.) media containing 1% (w/v) 2-(N-morpholino) ethanesulfonic acid (MES) (Duchefa Biochemie B.V.) with PH=5.8 and 0.8% Plant Agar (Lab M, MC029), and then stratified at 4  oC in dark for two days followed by pre-growth at 21 oC under 16 h light/8 h dark cycle and 65-70% humidity.  8-day-old seedlings were treated with 0 mM, 130 mM NaCl, 130mM NaNO3, 130mM KCl, 260mM Sorbitol or 25µM ABA for 6 h or 24h. Roots were dissected for harvest and stored into liquid N2 for RNA isolation. Approximately 40 roots were pooled into one biological replicate.  Total RNA was extracted using combined Tripure and RNA isolation kit (Qiagen). Total RNA was used for RNA library preparation suitable for Illumina HiSeq paired end sequencing using Illumina's TruSeq stranded mRNA kit for polyA mRNA selection. Then mRNA was processed directly including RNA fragmentation, first and second strand cDNA synthesis, adapter ligation and final library amplification according to the manufacturer's protocol. The final library was eluted in 30 µL elution buffer followed by quality assessment using a Bioanalyzer 2100 DNA1000 chip (Agilent Technologies) and quantified on a Qubit quantitation platform (Life Technologies).