RNA-seq of sugar beet: leaf, control at 25 degrees centigrade
Total RNAs were isolated with TRIzol reagent (Invitrogen, USA) and treated with TURBO DNase I (Ambion, USA) for 30 min to remove the DNA residues. Subsequently, RNA degradation and DNA contamination was supervised on 1% agarose gels. A Ribo-Zero rRNA Removal Kit (Epicentre, USA) was used for removing rRNA from 1.5 g RNA per sample. Sequencing libraries were prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biosystems, USA) following manufacturers recommendations, including synthesis of the first and second cDNA strands, end prep of DNA fragments, adaptor ligation, purification of DNA fragments with AMPure XP Beads (Beckman Coulter, Beverly, USA), PCR enrichment and purification of the PCR reaction. Index codes were also added to attribute sequences to each sample. Library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR. The index-coded samples was clustered by acBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS (Illumia) according to the manufacturers instructions. Last, an Illumina Hiseq platform was used for sequencing the libraries according to a 150-cycle paired-end sequencing protocol.