RNA sequencing of control and OsABCB24 overexpression mutant in leaf tissue samples (Oryza sativa japonica)
RNA sequencing of control and OsABCB24 overexpression mutant in leaf tissue samples (Oryza sativa japonica)
Leaf samples from three independent replicates of both WT and OsABCB24 overexpression lines were processed.  Dongjin cultivar of Oryza sativa subspecies japonica served as the wild type (WT). All transgenic lines being developed on the genetic background of Dongjin. Seedlings were germinated on Murashige and Skoog medium for 10 days and transferred to soil in a greenhouse or paddy field. The greenhouse conditions were maintained at 28°C with 16 hours of light and 22°C with 8 hours of darkness, and humidity of 70%.  Leaf samples were frozen directly in Liquid nitrogen and finely ground. Grounded samples were homogenized using TRI reagents (RNAiso, Takara) and BCP (1-bromo-3-chloropropane). DNA, RNA, and protein layers were separated by centrifugation. Then, total RNA was extracted using the RNeasy Mini Kit (QIAGEN). The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. Alternatively, \"tagmentation\" combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified.