title: GSM6744427: IR64-flag_leaf-BS-seq-1; Oryza sativa Indica Group; Bisulfite-Seq
library_construction_protocol: For collection of meiotic spikelets with meiocytes at prophase of meiosis I, tillers whose flag leaf auricle was about 3 cm under the auricle of the penultimate leaf were chosen and 0.4-0.6 cm long fresh spikelets were collected. Meiotic spikelets collected above were put on a clean slide. The anthers were gently dissected out and collected using dissecting needles under the dissection microscope.The anthers were put on a clean slide with 5 µl DEPC water/1× PBS added. Under an inverted microscope (with 10× objective), a 1 ml syringe needle was use to dissect one end of the anthers. Because the meiocytes at this stage produce callose, they stick together and form worm-like cell clusters. Gentle pressure was applied to the anthers to allow the separation of wormlike cell clusters from anther walls. The cell clusters were collected with capillary glass pipettes without taking any somatic cells. scBS-seq libraries were constructed using a reported protocol (Clark et al., 2017). The library was constructed using the CUT&Tag kit (Hyperactive® Universal CUT&Tag Assay Kit for Illumina, Vazyme Biotech Co.,Ltd).