RNA sequencing of control and OJAP_WD199 knock out mutant in mature rice anthers. (oryza sativa jap.)
RNA sequencing of control and OJAP_WD199 knock out mutant in mature rice anthers. (oryza sativa jap.)
The samples were collected from paddy field at anthesis stage. We froze the anthers immediately using liquid nitrogen. Plants were transplanted to paddy field after two weeks in incubator at BOD incubator at end of May. The plants were then grown for 3 months in the field. Mature anthers were frozen in LN2 and finely ground using mortar. 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.