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Sample GSM5135433 Query DataSets for GSM5135433
Status Public on Mar 08, 2022
Title MNaseSeq_WT_N3753
Sample type SRA
 
Source name nuclei from germinated condia
Organism Neurospora crassa
Characteristics sample type: nuclei
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. 
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 
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).
 
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model Illumina HiSeq 4000
 
Description MNase-seq
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
 
Submission date Mar 04, 2021
Last update date Mar 08, 2022
Contact name Hideki Tanizawa
E-mail(s) [email protected]
Organization name University of Oregon
Department Institute of Molecular Biology
Street address 1370 Franklin Blvd
City Eugene
State/province OR
ZIP/Postal code 97403
Country USA
 
Platform ID GPL23150
Series (1)
GSE168277 The ACF chromatin remodeling complex is essential for Polycomb repression
Relations
BioSample SAMN18141463
SRA SRX10240468

Supplementary file Size Download File type/resource
GSM5135433_WT_N3753_MNase_dyadCov.bw 32.7 Mb (ftp)(http) BW
GSM5135433_WT_N3753_MNase_specDensity.bw 2.5 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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