Strand-specific RNA-seq of RIL64 . Drought stressed .
Root total RNA were isolated using TRIzol® reagent (Invitrogen, USA) and treated with Dnase I (Rnase-free). RNA integrity was analyzed by a Tgilent Technologies 2100 Bioanalyzer with RNA integrity number (RIN) >8.0. The eight strand-specific RNA-seq libraries were constructed as described before with minor revisions (Hirsch et al. 2014). Briefly, mRNA were isolated and purified by OligoTex mRNA midi kit (Qiagen) and fragmented using the RNA fragmentation reagents (Ambion). The first strand cDNA was synthesized using random hexamer primers and second-strand cDNA was synthesized using a deoxynucleotide triphosphate mix wherein dUTP was chosed instead of dTTP. Double-stranded cDNA fragments were purified and ligated with adaptors. The second-strand cDNA were removed by dUTP digesting using AmpErase UNG (Applied Biosystems). RNA sequencing were performed at BGI-Shenzhen (Shenzhen, China) using Illumina deep sequencing according to the manufacturer's instructions.