RNA-Seq Library Preparation and Sequencing

A total of 5 ng RNA per sample was used as input material for sequencing libraries using the Ovation® SoLo RNA-Seq Library Preparation Kit (NuGEN, CA, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. For small RNA libraries, a total of 2.5 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR. The clustering of the index-coded samples was performed on acBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced using an Illumina Hiseq platform and paired-end reads were generated [25 (link), 26 (link)].