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| Status |
Public on Feb 15, 2024 |
| Title |
Cvip-55-IgG |
| Sample type |
SRA |
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| Source name |
seedling
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| Organism |
Oryza sativa Japonica Group |
| Characteristics |
chip antibody: IgG
treatment: hypermethylation
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| Treatment protocol |
For zebularine treatment, an inhibitor of DNA methyltransferase leading to a global reduction of DNA methylation, 100 of 5-d-old rice seedlings were treated with 80 µM zebularine (Sigma-Aldrich Z4775) dissolved in DMSO or DMSO only (as control) for another five days. The zebularine-treated or untreated seedlings were collected and kept at -80℃ until used. For genomic DNA with M.CviP I (M0227S, NEB) treatment, a type of CpG Methyltransferase resulting in a global increase CG methylation in the eukaryotic genomes. 4 μg of purified rice genomic DNA was incubated with 8 units of M.CviP I with an addition of 640 μM SAM (S-adenosyl-methionine) at 37℃ for 1 h in a total of 20 μl reaction system. After inactivation of the enzyme activity with incubation at 65℃ for 20 min, M.CviP I treated DNA was recovered by using phenol/chloroform extraction followed by pre-cold alcohol precipitation.
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| Growth protocol |
After pre-germination at room temperature (RT) for three days, uniformly germinated Nipponbare (Oryza sativa L., Japonica) rice seeds were put on the nutrient soil and grew in a greenhouse at 28-30℃ and a 14h/10h light-dark cycle. Two-week old seedlings were harvested and stored at -80℃ until used.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The fine powder was used for nuclei preparation and genomic DNA extraction following the procedures as described previously
The genomic DNA was fragmented into sizes, ranging from 100-500 bp, using the water-based Biorupter (Diagnode), followed by DNA extraction and purification. Total 5μg fragmented genomic DNA was diluted in iM stabilization buffer (50mM Tris-AcOH, pH=5.5) for denaturing and re-association treatment using a PCR program as below: 95°C for 8 min，95°C for 30s (-0.5 °C /cycles, 129 cycles)，35°C hold on. The re-associated DNA was diluted with iM-IP incubation buffer (50mM Tris-AcOH, 1mM MgCl2, 130nM CaCl2, 1%BSA and Complete mini, pH=5.5), then incubated with 3μg iMab antibody (Ab01462-23.0, Kappa) for 4 h at 4℃. The antibody incubation reaction was incubated with 30 μl of washed protein G Dynalbeads (10004D, Invitrogen) for another 4 h at 4°C. The iMab-bound DNA was finally eluted with 200 μl elution buffer (0.1M NaHCO3 and 1% SDS) at 65°C for two times with 15 min each. Three biologically replicated iM-IPed DNA or Input/IgG-IPed control DNA was used for library preparation. All libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645S). Libraries were finally quality controlled and sequenced using paired-end mode on Illumina NovaSeq platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
civ_pH55_merged_igg_peaks.narrowPeak
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| Data processing |
All clean reads in three biological replicates were aligned to the MSU v7.0 reference genome using BWA
SAMtools was used to exclude reads with mapping quality below 10
PCR duplicates were removed by using Picard
All aligned reads with at least length 50 were used for calling peaks by using callpeak function of MACS2 (version 2.1.1) (Zhang et al. 2008) with parameters as below: --extsize 200 -q 0.05 -nomodel -f BAMPE. Input and IgG data were used as controls.
Supplementary files format and content: narrowPeak files of MACS2 outputs
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| Submission date |
Jun 12, 2023 |
| Last update date |
Feb 15, 2024 |
| Contact name |
Xing Ma |
| E-mail(s) |
[email protected]
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| Organization name |
Nanjing Agricultural University
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| Street address |
No.1 Weigang, Nanjing, Jiangsu 210095, P. R. China
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| City |
Nanjing |
| ZIP/Postal code |
210095 |
| Country |
China |
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| Platform ID |
GPL27860 |
| Series (2) |
| GSE234751 |
Differential impacts of DNA methylation on pH dependent i-motifs formation in rice [ChIP-seq] |
| GSE234752 |
Differential impacts of DNA methylation on pH dependent i-motifs formation in rice |
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| Relations |
| BioSample |
SAMN35717481 |
| SRA |
SRX20662426 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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