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| Status |
Public on Aug 27, 2023 |
| Title |
B73 - MH-seq rep2 |
| Sample type |
SRA |
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| Source name |
B73 seedling
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| Organism |
Zea mays |
| Characteristics |
cultivar: B73
tissue: Leaf
age: 10 days
mnase concentration: 100 U MNase
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| Growth protocol |
Seeds of maize (Zea mays “B73”) were soaked in peri dish with wet paper towel and put at 23-25℃ for pregermination. Evenly germinated seeds were transferred to the pots containing soil and continued to grow in a growth chamber at a 16 h/8 h light-dark cycle at 25-28℃ for 10 days. Leaf tissues were collected from 10 individual 10-day-old plants for immediate downstream treatment or stored at -80 ℃ for later use.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
MH-seq: Two-week-old whole seedlings or small pieces cut from the 10-day-old leaf tissues (2-3 g) were soaked in fixation buffer (20mM HEPES, 1mM EDTA, 100mM NaCl, 1mM PMSF, pH 8.0) containing 1% of final concentration of formaldehyde (Sigma, cat. no. F-8775) under vacuum for 10 min at 23-25℃, then followed by adding 2 M Glycine (Sigma, cat. no. 50046) to a final concentration of 0.125 M under vacuum for additional 5 min for quenching the excessive formaldehyde. The cross-linked leaves were ground to a fine powder in liquid nitrogen. The ground powder was used for nuclei preparation and purification following published procedures. The purified nuclei pellet was resuspended in 1.2 ml of MNase digestion buffer (20 mM Tris-HCl, 4 mM MgCl2, 1mM CaCl2, 60 mM KCl, pH 7.5). The suspended nuclei were equally aliquoted into 5 of 1.5 ml Eppendorf tubes (200 μl each). The aliquoted nuclei were incubated in water bath at 37℃ for 10 min using a series of MNase (M0247, 2,000 gels units/μl, NEB) with a final amount as 0 unit (U), 90 U, 100 U, 700 U and 900 U, respectively, then followed by adding 16 μl 0.5 M EDTA (pH 8.0) to stop enzymatic reaction. Each MNase trimmed nuclei aliquot was added 16 μl 5 M NaCl, 8 μl 20% SDS and 160 μl 1X TE and incubated in 65℃ water bath overnight for reverse cross-linking. After sequential treatment using RNase A at 65℃ for 30 min and proteinase K at 55℃ for 2 h, reversed cross-linked nuclei were sequentially extracted by adding 1 volume of phenol, phenol:chloroform mixture and chloroform, then followed by pellet precipitation using 2 volumes of cold 200 proof ethanol. The air-dry DNA was dissolved using 20 μl EB (10 mM Tris-HCl, pH 8.0). The purified DNA was separated by running 2% of agarose gel in 1xTBE buffer. We chose DNA from 100 U and 900 U MNase-trimmed nuclei, representing the light and heavy MNase digestion condition, respectively, for recovering small-sized DNA fragments ranging from 40-100 bp (indicated by the red box in Fig. 1a).
MH-seq: The recovered small-sized DNA was used for MH-seq library preparation using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645S). The MH-seq library was quality controlled and sequenced using Illumina NovaSeq platform.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
B73-leaf-MH-seq-merged.bw
B73_MHS_merged_peaks.narrowPeak
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| Data processing |
Library strategy: MNase-seq
For MH-seq, MNase-seq and ChIP-seq data, sequencing reads were trimmed using fastp v0.20.0. Short reads (<50 bp) and reads with quality score (< 20) were removed, all trimmed reads were mapped to Zea mays B73 AGPv4 reference genome using Bowtie2. Aligned reads were sorted using SAMtools v1.5, properly paired reads were extracted using ‘samtools view -f 0x2 -q 20’.
Two biological MH-seq data were merged for MHS peak calling using MACS2 with ‘-f BAMPE -g 2.2e9 –nomodel -q 0.001 --fe-cutoff 5’. The R package ChIPseeker was used for MHS Peak annotation. L/HMNase-seq data were used for global identification of nucleosome positions. Nucleosome positions were identified by DANPOS2 using ‘dpos --paired 1 -p 1e-6 -q 0’. The highest point of each nucleosome was defined as the nucleosome center.
Genome_build: B73 v4
Supplementary_files_format_and_content: bigWig files were generated using deeptools bamCoverage.
Supplementary_files_format_and_content: narrowPeak files were generated using MACS2.
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| Submission date |
Aug 30, 2020 |
| Last update date |
Aug 27, 2023 |
| Contact name |
Wenli Zhang |
| E-mail(s) |
wzhang25@njau.edu.cn
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| Organization name |
Nanjing Agricultural University
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| Street address |
No.1, Weigang Street, Xuanwu District, Nanjing
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| City |
Nanjing City |
| ZIP/Postal code |
210095 |
| Country |
China |
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| Platform ID |
GPL25410 |
| Series (2) |
| GSE157149 |
Characterization of distinct types of open chromatin and their impacts on gene transcription in maize [MH-seq] |
| GSE157152 |
Characterization of distinct types of open chromatin and their impacts on gene transcription in maize |
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| Relations |
| BioSample |
SAMN15944579 |
| SRA |
SRX9038547 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4756687_B73-leaf-MH-seq-rep2.bw |
334.8 Mb |
(ftp)(http) |
BW |
| GSM4756687_B73_MHS_rep2_peaks.narrowPeak.gz |
1.6 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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