GSM3613364: ros1a-sperm; Oryza sativa; Bisulfite-Seq
Wild-type and ros1a/+ O. sativa cv. Nipponbare harboring a UBQ::H2B-GFP transgene were grown at 26 ºC in a 13/11 hr light/dark cycled chamber. Rice pollen was harvested from unopened flowers with elongated stamen. Pollen grains were burst open via osmotic pressure in 370 mOsmol/kg H2O mannitol solution for 5 minutes at room temperature. Sperm cells and vegetative cell nuclei were isolated using the differential interference contrast microscopy and GFP fluorescent microscopy, respectively, under a BX-71 inverted fluorescence microscope (Olympus, Tokyo, Japan). The sperm cells and vegetative cell nuclei were washed once in 370 mOsmol/kg H2O mannitol solution and stored at -80C. Bisulfite sequencing libraries were constructed as described Smallwood et al., Nature Methods (2014)