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| Status |
Public on Jun 24, 2023 |
| Title |
cmt3b-Zygote-3 |
| Sample type |
SRA |
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| Source name |
Zygote
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| Organism |
Oryza sativa Japonica Group |
| Characteristics |
cell type: Zygote
genotype: mutant-cmt3b
age: 6-8 hours after fertilization
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| Extracted molecule |
total RNA |
| Extraction 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
3b-Z-3
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| Data processing |
Raw reads of all sequencing data were trimmed using fastp (v0.20.1)
Clean read was then matched to the MSU7.0 rice reference genome using hisat2 software. FeatureCounts were used for quantitative analysis and the differentially expressed genes (Fold change > 2,q < 0.01) were calculated by DESeq2.
Clean reads were mapped to the MSU7.0 rice genome. Bismark(v0.22.3) software was used to match, deduplicate and extract methylation sites.
Supplementary files format and content: bigwig files for BS-seq, RNA-seq count files
Supplementary files format and content: bigwigs were derived from combining sample replicates and are linked to the first replicate
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| Submission date |
Jun 23, 2023 |
| Last update date |
Jun 24, 2023 |
| Contact name |
zhu bo |
| E-mail(s) |
[email protected]
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| Organization name |
Huazhong Agricultural University
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| Department |
National Key Laboratory of Crop Genetic Improvement
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| Lab |
National Key Laboratory
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| Street address |
shi zi shan street
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| City |
wuhan |
| State/province |
beijing |
| ZIP/Postal code |
430070 |
| Country |
China |
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| Platform ID |
GPL27860 |
| Series (1) |
| GSE235680 |
Function of CHG methylation dynamics in rice male gametogenesis |
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| Relations |
| BioSample |
SAMN35881009 |
| SRA |
SRX20763501 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7507610_cmt3b-Z-3.count.txt.gz |
698.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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