title: RNA sequencing of Nipponbare control and OsCNGC4, OsCNGC5 double knockout mutant in mature anthers
design: RNA sequencing of Nipponbare control and OsCNGC4, OsCNGC5 double knockout mutant in mature anthers
library_construction_protocol: Stages 13-14 anthers carrying tricellular stages pollen were collected. Seeds of japonica rice cv. Nipponbare were grown in the greenhouse for 4 weeks and transferred to the paddy field.  Mature anthers were frozen in LN2 and finely ground using Tissuelyser (QIAGEN). Then, total RNA was extracted using the MiniBEST Plant RNA Extraction Kit (Takakra, Japan) according to the manufacturerâ€™s instructions. 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.