ORE-seq: genome-wide absolute occupancy measurement by restriction enzyme accessibilities
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PROVIDER: PRJNA781633 | ENA |
REPOSITORIES: ENA
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Project description:Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1- accessibility) in absolute terms as the percentage of cut or uncut out of the total molecules. The here presented protocol takes this classical approach to the genome-wide level. After exhaustive restriction enzyme digestion of chromatin, DNA is purified, sheared and converted into libraries for high throughput sequencing. Bioinformatic analysis counts DNA fragments cut by the restriction enzyme as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straight forward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments with ends generated by restriction enzymes versus by shearing. Our protocol includes two orthogonal approaches to correct for this bias, the “corrected cut-uncut” and the “cut-all cut” method, so that accurate measurements of absolute accessibility/occupancy at restriction sites throughout a genome are possible. The protocol is presented for the example of S. cerevisiae chromatin but may be adapted for any other species.
2023-01-01 | GSE189142 | GEO
Project description:Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome [ORE-seq]
| PRJNA591902 | ENA
Project description:Mapping of nucleosomes, the basic DNA packaging unit in eukaryotes, is fundamental for understanding genome regulation as nucleosomes modulate DNA access by their positioning along the genome. A cell population nucleosome map requires two observables: nucleosome positions along the DNA (“Where?”) and nucleosome occupancies across the population (“In how many cells?”). All available genome-wide nucleosome mapping techniques are yield methods as they score either nucleosomal (e.g., MNase-seq, chemical cleavage-seq) or non-nucleosomal (e.g., ATAC-seq) DNA but lose track of the total DNA population for each genomic region. Therefore, they only provide nucleosome positions and maybe compare relative occupancies between positions but cannot measure absolute nucleosome occupancy, which is the fraction of all DNA molecules occupied at a given position and time by a nucleosome. Here, we established two orthogonal and thereby crossvalidating approaches to measure absolute nucleosome occupancy across the Saccharomyces cerevisiae genome via restriction enzymes and DNA methyltransferases. The resulting high-resolution (9 bp) map shows uniform absolute occupancies. Most nucleosome positions are occupied in most cells: 97% of all nucleosomes called by chemical cleavage-seq have a mean absolute occupancy of 90 ± 6% (± SD). Depending on nucleosome position calling procedures, there are 57-60,000 nucleosomes per yeast cell. The few low absolute occupancy nucleosomes do not correlate with highly transcribed gene bodies, but with increased presence of the nucleosome-evicting RSC chromatin remodeling complex there and are enriched upstream of highly transcribed or regulated genes. Our work provides a quantitative method and reference frame in absolute terms for future chromatin studies.
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2009-10-08 | GSE13744 | GEO
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2009-10-27 | GSE15835 | GEO
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Project description:Time course of batch growth. Reference (channel 1) was a culture grown in MD medium with 2.4 g/L glucose, with 5 slpm air-flow, stirring at 400 rpm and a constant 300C temperature, dilution 0.25 volumes/hour. The experimental (channel 2) samples were grown in batch for the indicated time. Groups of assays that are related as part of a time series. FactorCategory: Elapsed Time; name: Elapsed Time; Measurement: time(absolute) 6 h; name: 34357_Elapsed Time; Measurement: time(absolute) 10.5 h; name: 34365_Elapsed Time; Measurement: time(absolute) 8 h; name: 34358_Elapsed Time; Measurement: time(absolute) 9 h; name: 34359_Elapsed Time; Measurement: time(absolute) 11 h; name: 34368_Elapsed Time; Measurement: time(absolute) 9.5 h; name: 34360_Elapsed Time; Measurement: time(absolute) 10 h; name: 34362_Elapsed Time Keywords: time_series_design
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