Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea

Transcriptome sequencing performed at the Novogene Bioinformatics Institute (Novogene, Beijing, China). Briefly, mRNA was purified from the total RNA of tea leaves (Camellia assamica var. kucha and Camellia sinensis var.assamica) and used to construct sequencing libraries using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations. The index codes were added to attribute sequences to each sample. After the qualities of sequencing libraries were confirmed on the Agilent Bioanalyzer 2100 system, clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, CA, USA) according to the manufacturer’s instructions. The library preparations were then sequenced on an Illumina Hiseq 2500 platform (Illumina, San Diego, CA, USA) and paired-end reads were generated. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity with min_kmer_cov set to 2 by default and all other parameters set default.

本研究的转录组测序（Transcriptome sequencing）工作在中国北京诺禾致源生物信息研究所（Novogene）完成。简言之，实验人员从茶树（Camellia assamica var. kucha与Camellia sinensis var. assamica）叶片的总RNA中纯化得到信使RNA（mRNA），随后参照美国纽英伦生物科技（NEB, USA）的NEBNext® Ultra™ RNA Library Prep Kit for Illumina®试剂盒说明书的操作流程构建测序文库，并添加索引序列以区分不同样本。在使用安捷伦生物分析仪2100系统（Agilent Bioanalyzer 2100 system）验证测序文库质量后，实验人员参照美国加利福尼亚州圣地亚哥Illumina公司的说明书，采用TruSeq PE Cluster Kit v3-cBot-HS试剂盒，在cBot集群生成系统上完成索引标记样本的集群扩增。随后将制备好的文库置于Illumina HiSeq 2500测序平台（Illumina, San Diego, CA, USA）进行测序，生成双端reads数据。转录组组装工作基于left.fq与right.fq双端测序数据，使用Trinity软件完成，其中min_kmer_cov参数默认设置为2，其余参数均采用默认配置。