Agapanthus praecox subsp. orientalis strain:Big Blue Transcriptome or Gene expression. Agapanthus praecox subsp. orientalis strain:Big Blue

In order to obtain more comprehensive transcriptomic information of Agapanthus, 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.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).

为获取更全面的百子莲（Agapanthus）转录组信息，本研究采集了叶片、根系、块茎、茎尖及胚性愈伤组织等多种器官的等量样本。采用TRIzol试剂（Invitrogen，中国上海）依照产品说明书提取混合组织的总RNA。使用无RNase脱氧核糖核酸酶I（RNase-free DNase I，TaKaRa，日本滋贺县大津市）在37℃下处理总RNA 30分钟，以去除残留的基因组DNA。从样本中分离得到总RNA后，利用寡聚(dT)磁珠（Oligo (dT) linked beads）富集聚腺苷酸信使RNA（poly (A) mRNA）。通过片段化缓冲液将mRNA切割为短片段，以此作为模板，采用随机六聚体引物合成第一链互补脱氧核糖核酸（cDNA）。随后使用缓冲液、脱氧核糖核苷三磷酸（dNTPs）、核糖核酸酶H（RNase H）及DNA聚合酶I合成第二链cDNA。依照产品说明书，使用基因组样本制备试剂盒（Genomic Sample Prep kit，Illumina）构建双端测序文库。采用QIAquick PCR提取试剂盒（Qiagen）纯化短片段，再以EB缓冲液洗脱以进行末端修复并添加poly (A)尾。将短片段与测序接头连接后，通过琼脂糖凝胶电泳筛选符合长度要求的片段。最终通过PCR扩增完成测序文库构建，并采用HiSeqTM 2000测序平台（Illumina）完成测序。