OsRALF17/19-3
Oryza sativa Japonica Group
Protocols: Anthers at the bicellular and tricellular stages of stages 11-13 were collected. Seeds of japonica rice cv. Dongjin was sterilized with 50% of sodium hypochlorite and germinated in MSO media in an incubator (14-h light/10-h dark, 28 °C (day)/22 °C (night), humidity 80%). After 1 week of germination, seedlings were transferred and grown in the greenhouse for a period of 4 weeks under growth chamber conditions and transferred to 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.