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| Status |
Public on Apr 12, 2024 |
| Title |
CHA4_Mutant_MNase |
| Sample type |
SRA |
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| Source name |
Yeast cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell type: Yeast cells
strain: BY4742
genotype: cha4{delta}
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| Growth protocol |
Yeast cells were grown in yeast extract peptone dextrose (YEPD) medium to an OD of 0.6-0.8
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cultures were cross-linked with 1% formaldehyde at room temperature (RT) for 30 min. and quenched with 0.125 M glycine at RT for 5 min. to neutralize the formaldehyde. Cells were centrifuged, washed withwater, and treated with Buffer Z (0.56M sorbitol, 50mM Tris) with 0.5 mL of 10mg/ml zymolyase at 25°C for 30 min. Extracts were centrifuged at 1500 rpm for 6 min. at 4°C and resuspended in NP buffer (1M sorbitol, 50mM NaCl, 10mM Tris pH 7.4, 5mM Mgcl2, 1mM CaCl2) containing 250mM spermidine, 0.007% b-ME, and 0.075% NP-40. In separate tubes, MNase was aliquoted as follows: 4 µL, 2 µL, 1 µL, 2 µL of 1:4 in water, and 1 µL in 1:4 water. 400 µL of extract were added to each MNase aliquot and incubated for 20 min. at RT. After digestion, 100 µL stop buffer (5mM SDS, 1mM EDTA) and 10 µL of 10mg/ml Proteinase K were added. Samples incubated O/N at 65°C. To recover DNA, samples were phenol:chloroform extracted and precipitated with 400 µL of isopropanol. DNA was treated with 0.1mg/ml RNaseA to remove RNA.
Illumina sequencing libraries of MNase-treated DNA were prepared using 500 ng of DNA as previously described (Henikoff et al 2011).
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
DM930
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| Data processing |
FASTQ reads were aligned to sacCer3 (Saccharomyces cerevisiae) using bowtie v1.1.1 (Langmead 2010) using the paired-end option with the following bowtie flags: -m1 -n 2 -l 20 --best --strata -S - y --phred 33 --nProc 32. Aligned files were processed and converted into BAM format using samtools v1.2 (Heng Li et al. 2009).
Overall chromatin, nucleosome, and TF occupancy changes were quantified by calculating differences between fixed windows across every gene in the genome between control and mutant. Specific methods are described in the methods section of the paper.
Assembly: sacCer3, R64
Supplementary files format and content: RDS files containing Jensen-Shannon divergence, transcription factor, and nucleosome occupancy quantifications for each mutant. Specific details of the analyses are described in the Methods section of the manuscript
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| Submission date |
Apr 06, 2024 |
| Last update date |
Apr 26, 2024 |
| Contact name |
David MacAlpine |
| E-mail(s) |
david.macalpine@duke.edu
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| Organization name |
Duke University
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| Department |
Department of Pharmacology and Cancer Biology
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| Street address |
308 Research Dr, Durham, NC 27710
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE263367 |
Genome-wide nucleosome and transcription factor responses to genetic perturbations reveal mechanisms of chromatin-mediated transcriptional regulation |
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| Relations |
| BioSample |
SAMN40848056 |
| SRA |
SRX24173768 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8190310_CHA4_quantifications_length_corrected.RDS.gz |
662.1 Kb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
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