RNA sequencing of control and OsADF1/6/9 triple knockout mutant in mature rice anthers (Oryza sativa japonica)
RNA sequencing of control and OsADF1/6/9 triple knockout mutant in mature rice anthers (Oryza sativa japonica)
Anthers that include bicellular and tricellular stages of pollen were collected (11 to 13 stages of Anthers). Seeds of japonica rice cv. Dongjin was sterilized with 50% sodium hypochlorite and germinated in MS zero 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 4 weeks under growth chamber conditions and transferred to the field.  Mature anthers were frozen directly in Liquid nitrogen 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.