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| Status |
Public on Mar 08, 2022 |
| Title |
MNaseSeq_crf4-2_N8016 |
| Sample type |
SRA |
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| Source name |
nuclei from germinated condia
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| Organism |
Neurospora crassa |
| Characteristics |
sample type: nuclei
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| Treatment protocol |
Cells were crosslinked by addition of fresh formaldehyde for a final concentration of 1%, then shaken at room temperature for 10 minutes. The crosslinking reaction was quenched by the addition of 6.8 ml of 1M Tris-HCl pH 8 to a final concentration of 125mM for 10 minutes, shaking at room temperature. Conidia were then harvested by centrifugation.
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| Growth protocol |
A 50 ml culture of Vogel’s solution plus 1.5% sucrose was inoculated with 2.5E7 cells, harvested from freshly grown conidia, and grown at 32oC with shaking for ~3.5 hours, until at least 70% of conidia have germinated as determined by microscopy
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated with 2 mg of zymolyase (100T) in a 1 ml solution of spheroplast buffer (1M sorbitol, 50mM Tris pH 7.5, 10mM BME) for 15 minutes while rotating at room temperature. Cells were pelleted at full speed and resuspended in 100 ul MNase digestion buffer (1M sorbitol, 50mM NaCl, 10mM Tris pH 7.5, 5mM MgCl2, 1mM CaCl2, 0.075% (v/v) IGEPAL (Sigma), 0.5mM spermidine and 1mM BME). Exonuclease III (NEB) (30 units) and micrococcal nuclease (Takara) were added to samples and rapidly mixed. Digestion proceeded for 10 minutes at 37oC and was quenched with final concentration of 5mM EGTA and 5mM EDTA (from 50mM/50mM stock). MNase concentration was optimized for each strain to yield ~80-90% mononucleosomes (20 units for N3752, N3753, N7966, N8018, N7990, N7992, N7988, N7989; 60 units for N4718; and 80 units for N4730, N6170, N6171, N8016, N8017). RNase (40 ug) treatment was for 1.5 hours at 42C and proteinase K (200 ug) treatment was for 1 hour at 65 C.
10 ug of gel-purified (minelute, Qiagen) mononucleosome DNA was prepared for high-throughput sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
MNase-seq
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| Data processing |
Paired-end sequence reads were aligned to the neurospora crassa genome (OR74A) using Bowtie2 (version 2.3.3) with the option “-q -p 4 -X 250 --no-discordant --no-mixed --no-unal” (Paired-end alignment reads with maximum 250bp distance gap between them were used in subsequent analysis. This length correspond to mono-nucleosome)
Only correctly aligned paired-end alignment reads were filtered using samtools (version 1.5) commands “samtools view –hf 0x2 input.bam | grep –v “XS:i:” “
Dyad Coverage was calculated using the scripts (03_PNA_SDE.R) from Baldi et al (Baldi, S. et al. Genome-wide Rules of Nucleosome Phasing in Drosophila. Mol. Cell 72, 661-672.e4 (2018))
The spectral density (SD) score correspond to periods of 182bp was calculated using the scripts (cov2spec.R) from Baldi et al. SD score was normalized as Z-score : (log2(SD score) – average) / standard deviation.
Regions with the average Z-score threshold of 2 were defined as the domain of a regular nucleosome array.
neurospora crassa genome (OR74A)
bigwig of dyad coverage and spectral density
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| Submission date |
Mar 04, 2021 |
| Last update date |
Mar 08, 2022 |
| Contact name |
Hideki Tanizawa |
| E-mail(s) |
[email protected]
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| Organization name |
University of Oregon
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| Department |
Institute of Molecular Biology
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| Street address |
1370 Franklin Blvd
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| City |
Eugene |
| State/province |
OR |
| ZIP/Postal code |
97403 |
| Country |
USA |
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| Platform ID |
GPL23150 |
| Series (1) |
| GSE168277 |
The ACF chromatin remodeling complex is essential for Polycomb repression |
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| Relations |
| BioSample |
SAMN18141480 |
| SRA |
SRX10240479 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5135444_crf4-2_N8016_MNase_dyadCov.bw |
32.9 Mb |
(ftp)(http) |
BW |
| GSM5135444_crf4-2_N8016_MNase_specDensity.bw |
2.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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