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| Status |
Public on Jul 01, 2021 |
| Title |
G4-ChIP-seq_K++PEG_rep1 |
| Sample type |
SRA |
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| Source name |
Seedlling
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| Organism |
Oryza sativa |
| Characteristics |
cultivar: Nipponbare
tissue: Seedling
age: two weeks
antibody: anti-FLAG antibody (D110005, BBI)
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| Growth protocol |
Rice (Oryza. sativa L. spp. japonica) seeds were immerged in tap water at RT in the lab for three days. Uniformly germinated seeds were sowed in the nutrient soil and grown in a greenhouse at a temperature at 28-30 ℃ and a 14 h/10 h light-dark cycle. Two-week-old rice seedlings were collected and cross-linked using 1% of formaldehyde for 10 min under vacuum. The cross-linked seedlings were ground into a fine powder in liquid nitrogen, and stored in -80 ℃ or immediately used for preparation of genomic DNA.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The purified nuclei were fragmented into sizes ranging from 100-500 bp in sonication buffer (50 mM Tris-HCl + 10 mM EDTA + 1% SDS) using the water-based Biorupter (Diagnode) followed by reverse cross-linking at 65 ℃ overnight，which 5M NaCl was added to a final concentration 0.2M. The reverse cross-linked DNA was purified by phenol:chloroform extraction and ethanol precipitation.Total 5 μg fragmented genomic DNA was diluted in G4 stabilization buffer (GSB) (150 mM KCl + 40% PEG200 + 10 mM Tris-HCl, pH=7.5), and denatured at 95 ℃ for 5 min followed by reassociation with the temperature slowly dropped down to RT. The reassociated DNA was diluted with G4-ChIP incubation buffer (GCB) (50 mM HEPES + 150 mM KCl+ 1 mM MgCl2+ 130 nM CaCl2+1 % BSA+ 40% PEG200 + Complete mini, pH 7.5). 3 μg purified BG4-FLAG recombinant protein was added and incubated at 4 ℃ for 4 h with constant rotation. 3 μg anti-FLAG antibody (D110005, BBI) was then added and incubated at 4 ℃ for additional 4 h. 30 μl protein G Dynalbeads (10004D, Invitrogen) was washed with incubation buffer for three times, and added to the reaction for incubation at 4℃ for another 4 h. After protein G beads incubation, the beads were collected and washed with washing buffer (10 mM Tris-HCl + 150 mM KCl + 1% Tween20) for three times. The BG4-bound DNA was eluted with 200 μl elution buffer (0.1 M NaHCO3 + 1% SDS) at 65 ℃ for two times with 15 min each. ChIPed and corresponding input DNA was recovered and dissolved in 20 EB (10 mM Tris-HCl, pH=8.0). For BG4-ChIP-seq assay, the BG4-ChIPed DNA was used for library preparation for Illumina sequencing followed by data analysis.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw G4-ChIP-seq data were cleaned through trimming adapter contamination and removing low-quality reads using Trim Galore! (version 0.4.4). All cleaned reads were aligned to the MSU v7.0 reference genome using BWA (Burrows-Wheeler Aligner) (mem algorithm, version 0.7.17) with default parameters. Any PCR duplicates were removed using Picard. Reads with mapping quality below ten were excluded using SAMtools (version 1.5, option -q). Only reads with alignment length greater than 50 were used for G4 peak calling and further analyses.
G4 Peak calling was performed using MACS (version 2.1.1) with the following command and parameters: macs2 callpeak -g 3.8e+8 -f BAM --extsize 200 -p 1e-5 –nomodel, and the input library was used as control.
The PQS loci file were generate using fastaRegexFinder.py through the rice7 MSU genome
G2 pattern were identified using regular expression ([gG]{2}\w{1,12}){3,}[gG]{2}
G3 pattern were identified using regular expression ([gG]{3}\w{1,12}){3,}[gG]{3}
Genome_build: Rice MSU v7.0
Supplementary_files_format_and_content: wig files were generated using bamCoverage and bigWigToWig, bed files were generated using macs2
Supplementary_files_format_and_content: xlsx files with PQS loci for each chromosome
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| Submission date |
Jun 14, 2019 |
| Last update date |
Jul 01, 2021 |
| Contact name |
Pengyue Zhang |
| E-mail(s) |
zhangpy195@163.com
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| Organization name |
NanJing Argriculture University
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| Street address |
No.1, Weigang Street, Xuanwu District, Nanjing
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| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
210095 |
| Country |
China |
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| Platform ID |
GPL19290 |
| Series (1) |
| GSE132775 |
Characterization of G-quadruplexes in the rice genome |
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| Relations |
| BioSample |
SAMN12060231 |
| SRA |
SRX6073410 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3891763_G4_K_PEG_ChIP_rep1.wig.gz |
106.9 Mb |
(ftp)(http) |
WIG |
| GSM3891763_G4_K_PEG_ChIP_rep1_peak.bed.gz |
1.1 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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