title: de novo transcriptome sequencing of Agapanthus praecox
design: we collected equal amounts of various organs including leaf, root, stem tuber, stem tip and embryogenic callus. Total RNA of mixed tissue was isolated using TRIzol reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Total RNA was treated with RNase-free DNase I (TaKaRa, Otsu, Shiga, Japan) for 30 min at 37°C to remove residual DNA. The RNA integrity (RNA Integrity Score is 7.9) and quantity were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies) before cDNA synthesis.  Oligo (dT) linked beads were used to isolate poly (A) mRNA after total RNA had been collected from the samples. Fragmentation buffer was used to chop the mRNA into short fragments, which were then used as templates for random hexamer-primed synthesis of first-strand cDNA. Second-strand cDNA was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I. The paired-end library was synthesized using the Genomic Sample Prep kit (Illumina), according to the manufacturer’s instructions. Short fragments were purified with the QIAquick PCR (Qiagen) extraction kit and then resolved with EB buffer for end repair and the addition of poly (A). The short fragments were then connected with sequencing adapters, and suitable fragments were separated by agarose gel electrophoresis. Finally, the sequencing library was built by PCR amplification and sequenced using the HiSeqTM 2000 platform (Illumina).