title: GSM7507560: BS-ZH11-Sperm-2; Oryza sativa Japonica Group; Bisulfite-Seq
library_construction_protocol: Rice sperms, meiocytes and unicellular microspores were isolated from anthers of Cas9-free transgenic plants and tissue culture-regenerated wild type plants. For sperm isolation, mature anthers were soaked in 45% (w/v) sucrose and then transferred into 15% (w/v) sucrose to release sperm pairs. For isolation of meiocytes, panicles of about 5 cm length were chosen and middle parts of the panicles were collected. Intact anthers were carefully selected using dissecting needles, placed onto an RNAase-free slide, and suspended in a PBS solution containing 1% proteinase inhibitors. The outer wall of anthers was then removed by capillary to release meiocytes. For isolation of unicellular microspores, panicles of about 8 cm length were harvested and the middle parts of the panicles were collected. Anthers were dissected out and make a hole with a capillary tube in the same solution as used for meiocyte isolation. Squeeze gently from the opposite side of the hole to release unicellular microspore. All isolated cells were collected by a micromanipulator system (Eppendorf, TransferMan® 4r).Rice eggs and unicellular zygotes were isolated from ovules before and after fertilization of Cas9-free transgenic plants and tissue culture-regenerated wild type (Zhou et al., 2019; Zhou et al., 2021). Briefly, ovaries of non-pollinated and pollinated florets (about 6.5 h after pollination) were manually collected in RNAase-free water under a dissection microscope. The collected ovules were transferred to a solution containing 0.53 M mannitol and 1% proteinases inhibitors to release eggs or zygotes. All isolated cells were stained by FDA (Fluoresceinc diacetate, Sangon, 596-09-8) and collected by a micromanipulator system (Eppendorf, TransferMan® 4r). For RNA-seq library construction, mRNA isolated from different types of cells were reverse transcribed and amplified by utilizing SMART-Seq® v4 Ultra® Low Input RNA Kit (TAKARA, Cat. No.634889). cDNAs were sheared into 200-400 bp DNA fragments followed by purification using Agencourt AMPure Beads (Beckman Coulter, USA A63881). The rest steps were performed using the Truseq ChIP DNA library preparation kit (Illumina. IP-202-1012). About 3000 sperm cells and 400 meiocytes were used to construct the transcriptome libraries. Fifty cells of egg or unicellular zygote were used for each replicate of the transcriptome library construction. BS-seq libraries were constructed using reported protocol (Clark et al., 2017). About 300-400 cells were pooled for each replicate for sperm, meicoyte and unicellular microspore bisulfite seq library construction and fifty cells of egg or unicellular zygote were used to construct bisulfite seq library.