After each step, seeds were gently washed with distilled water three times. All procedures were performed aseptically

in a laminar hood. To induce adventitious roots, cotyledons separated from sterilized stratified seeds were cultured on a solid Schenk and Hildebrandt (SH) medium containing 2.0 mg/L indole butyric acid, 3% sucrose, and 0.23% Gelrite. After 1 month, induced adventitious A 1210477 roots were separated from cotyledon explants and cultured again for secondary growth on the same medium. Then, the roots were transferred to a 30 mL liquid SH medium supplemented with 3.0 mg/L indole butyric acid and 5% sucrose, and maintained on a rotary Selleckchem Idelalisib shaker (100 rpm) at 25°C in the dark. For further mass production, 12 g fresh adventitious roots in suspension culture were inoculated into a 2 L airlift balloon-type bioreactor (Biopia, Korea) containing 1 L of the same SH medium as that used for liquid suspension culture (Fig. 1). The medium was replaced with a fresh medium after 2 weeks, and 4 weeks

later, 12 g adventitious roots were subcultured into a new bioreactor. After 10 days of cultivation, the subcultured adventitious roots were used for total RNA extraction with the Plant RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Approximately 2 μg total RNA from each cultivar was used for sequencing on the Illumina platform after the quality and quantity were checked using spectrophotometry. Paired-end reads with an average

length of 101 bp were generated for CP and CS using the Illumina Hiseq2000 platform. Library construction and sequencing were performed by the National Instrumentation Center and Environmental Management (NICEM), Seoul National University, Seoul, South Korea. Protirelin The sequence data generated in this study have been deposited in the Short Read Archive (SRA) of the NCBI under the accession number SRA061905. The sequencing reads underwent various stringent quality controls, such as filtering of high-quality reads and removal of reads with an adaptor or primer-contaminated sequence using the NGS QC toolkit [17]. All de novo assemblies were performed on a server with 48 cores and 512 GB random access memory. Publicly available transcriptome and genome assemblers were used to assemble the paired-end reads. Among the transcriptome assemblers, the open source program, Oases [18] (version: 0.2.06; http://www.ebi.ac.uk/∼zerbino/oases), which uploads a preliminary assembly produced by Velvet, was validated for k-mer optimization. Various assembly parameters were also examined to yield statistically as well as biologically significant results.