title: RNA sequencing of control and OsABCG16, OsABCG28 multiple knockout mutants in mature rice anthers (Oryza sativa jap.)
design: RNA sequencing of control and OsABCG16, OsABCG28 multiple knockout mutants in mature rice anthers (Oryza sativa jap.)
library_construction_protocol: Mature anthers that include bicellular and tricellular stages of pollen were collected (11 to 13 stages of Anthers). As this transgenic line exhibits complete sterility and fails to produce seeds, the plants analyzed in this study were T0 lines generated through tissue culture. These seedlings were maintained under growth chamber conditions in a greenhouse for four weeks prior to their transplantation into the paddy 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.