Full-length Transcriptome Analysis of Artemisia tangutica Endemic to Qinghai-Tibetan Plateau

The fresh and tender leaves of Artemisia tangutica for sequencing were collected from Wulan Xian, Mongolian and Tibetan Autonomous Prefecture of Haixi, Qinghai Province (geographic coordinates: N 36 ° 56'37 ", E 98 ° 27'20"; altitude: 2939 meters). After the leaves were collected, they were quickly stored in liquid nitrogen. The voucher specimen (chen2019104) was deposited in the Qinghai Tibet Plateau Biological Herbarium (HNWP) of the Northwest Plateau Institute of Biology, Chinese Academy of Sciences. The total RNA of Artemisia tangutica leaves was extracted by Trizol method. Agarose gel electrophoresis was used to check whether the extracted RNA was contaminated, and the RNA concentration was detected by nanodrop. The quality and integrity of RNA were evaluated by nanophotometer spectrophotometer and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). For qualified RNA samples, Oligo DT was used as primer to reverse the target mRNA, and full-length cDNA was amplified by low cycle PCR. The NEBNext End repair/dA tailing Module is used for terminal repair and A addition, and the ONT SQKLSK109 kit and NEBNext Quick Ligation Module are used for connection of sequencing connector. The sequencing platform is PromethION (Oxford Nanopole Technologies, UK).

用于测序的唐古特蒿（Artemisia tangutica）鲜嫩叶片采集自青海省海西蒙古族藏族自治州乌兰县，地理坐标为北纬36°56′37″、东经98°27′20″，海拔2939米。叶片采集后迅速置于液氮中保存。凭证标本（chen2019104）存放于中国科学院西北高原生物研究所青藏高原生物标本馆（HNWP）。采用Trizol法提取唐古特蒿叶片总RNA，通过琼脂糖凝胶电泳检测所提取RNA的污染情况，利用nanodrop检测RNA浓度，并通过纳米分光光度计（nanophotometer）与安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer，Agilent Technologies，美国加利福尼亚州帕洛阿尔托）评估RNA的质量与完整性。对于合格的RNA样品，以Oligo DT为引物反转录目标mRNA，并通过低循环PCR扩增获得全长cDNA。使用NEBNext末端修复/dA加尾模块完成末端修复与A尾添加，再利用ONT SQK-LSK109试剂盒与NEBNext快速连接模块完成测序接头的连接。测序平台为PromethION（英国牛津纳米孔科技公司）。