GSM9573748: ZH11-BM-H3K27me3-2; Oryza sativa Japonica Group; OTHER
For collection of tetrads and microspores, when tillers flag leaf collar and penultimate leaf collar aligned spikelets of 0.4-0.6 cm length were taken. Like extracting meiocytes, anthers were dissected out and gentle pressure was applied to allow the release of germ cells. Cells with tetrad shapes were collected as tetrads. Cells with single, free-floating state and have not formed cell walls were collected as microspores.For collection of mature unicellular microspores, when tillers flag leaf auricle was about 5 cm above the auricle of the penultimate leaf, fresh spikelets > 0.6 cm were collected. At this stage the color of the spikelets is still white. Anthers were dissected out and gentle pressure was applied to allow the release of germ cells. Cells with distinct germination pores and having formed cell walls are collected as unicellular microspores.For collection of bicellular microspores, spikelets turning greener in color were taken. Anthers were dissected out and gentle pressure was applied to allow the release of germ cells. Cells with distinct large vacuoles, two nuclei, and thickened pollen walls are collected as bicellular microspores.For collection of mature pollen, tillers green spikelets were taken. At this point, the anthers have developed into yellow color. Anthers were dissected out and gentle pressure was applied to allow the release of germ cells. The cells that are distinctly filled with starch are collected as pollen.A differential centrifugation isolation method was used to separate sperm from whole rice anthers. Isolation and purification of mRNA from different types of cells were performed using the Single Cell Full Length mRNA Amplification Kit (Vazyme, Cat. # N712). The RNA-seq libraries were prepared using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, Cat. # TD502) and sequenced on Illumina HiSeq 6000. For rice in SY63 backgrounds, we used single male germ cell to construct the transcriptome library. We used approximately 100-200 male germ cells to construct the transcriptome library.BS-seq libraries were constructed using reported protocol Clark et al (2017). About 400–500 male germ cells were pooled for each replicate bisulfite seq library construction.The CUT&Tag libraries were prepared using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, Cat. # TD903) and sequenced on Illumina HiSeq 6000. We used approximately 10,000 male germ cells to construct the CUT&Tag library by male germ cell isolation technology. The male germ cells were lysed with 200 µL Nuclear extraction buffer 2 (NEB2) (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100, 1× protease inhibitor), and the nuclei pellet was resuspended in 100 µL wash buffer (from the TD903 kit) for library construction. About 100,000 sperm cells were pooled for each replicate CUT&Tag library construction. About 100,000–200,000 seedling cell nuclei were pooled for each replicate CUT&Tag library construction.For ATAC-seq library construction, the Hyperactive ATAC-Seq Library Prep Kit for Illumina (Vazyme, Cat. # TD711) was used to prepare libraries for different types of cells. Using the same number of cells as for CUT&Tag, we isolated approximately 10,000 male germ cells to construct the ATAC-seq library. Approximately 100,000 sperm cells and 100,000–200,000 seedling cell nuclei were pooled for each replicate ATAC-seq library construction.