RNA preparation, Illumina paired-end cDNA library construction and sequencing

Total RNA was extracted from each tissue using the TRI reagent (Sigma-Aldrich, St. Louis, USA). RNA integrity was confirmed by agarose electrophoresis, and RNA quantification was performed using a Nanodrop Spectrophotometer ND-1000 (Thermo Scientific, Wilmington, USA). For each of the two accessions, we combined equivalent amounts of RNA from each tissue into two pools. A total of 10 μg of total RNA for each pool was sent to Macrogen Korea (Seoul, South Korea) for Illumina RNA-seq performed in HiSeq 2000 sequencer (Illumina, San Diego, USA).

The cDNA library was constructed according to the manufacturer’s instructions (Illumina/Hiseq-2000 RNA-seq) by Macrogen Korea. Essentially, the mRNA molecules containing poly (A) were purified using Sera-mag Magnetic Oligo (dT) Beads from the RNA samples. A fragmentation buffer was added to break the mRNA into small fragments. Using these fragments as templates, the first strand of cDNA was synthesized. The second strand of cDNA was synthesized using the buffers containing dNTPs, RNase H, and DNA polymerase I. The synthesized cDNA was purified and connected with the sequencing adapters. Finally, a range of cDNA fragments (200 ± 25 bp) were excised from an agarose gel using a gel extraction kit. Then, the library was sequenced using the Illumina/Hiseq-2000 RNA-seq. These raw sequences are available at the NCBI Sequence Read Archive (SRA) as stated in the section titled, “Availability of Data and Material”.