mRNA sequencing of Nicotiana benthamiana. RNA-seq-NI

Experiments designed and performed by Martin H. Schattat and Jessica L. Erickson (Institute of Plant Physiology, Martin Luther University Halle-Wittenberg, Germany). Three transgenic N. benthamiana plants were grown on soil under 8h light (120μEm^{−2} ) and 16h dark at 20° C. The transgene, an expression cassette facilitating the expression of a stroma targeted eGFP, was introduced into N. benthamiana for the purpose of examining the plastids. A total of approximately 100mg of plant tissue was harvested from the 3rd and 4th leaves of six week old plants. The Qiagen RNeasy Plant Mini Kit was used to isolate total RNA from the three plants (three biological replicates), which was then sent to MWG Eurofins (Ebersberg, Germany) for RNA sequencing with Illumina HiSeq 2000 (v3.0 chemistry).

本实验由德国马丁路德哈勒-维滕贝格大学植物生理学研究所的Martin H. Schattat与Jessica L. Erickson设计并开展。研究所用3株转基因本氏烟草（N. benthamiana）种植于土壤中，培养条件为20℃下，每日接受光照强度120 μmol·m⁻²·s⁻¹的光照8小时，随后保持16小时黑暗。本次实验转入的外源基因为一段表达盒，可介导靶向叶绿体基质的增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）的表达，实验通过构建该转基因株系以研究质体。待植株生长至6周龄时，从其第3、4片叶片采集总量约100 mg的植物组织。采用Qiagen RNeasy植物总RNA提取试剂盒，从3株植株（设置3个生物学重复）中分离总RNA；随后将总RNA寄送至德国埃伯斯贝格的MWG Eurofins公司，使用Illumina HiSeq 2000（v3.0 测序化学试剂）完成RNA测序。