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| Status |
Public on Jan 01, 2023 |
| Title |
ORE-seq: genome-wide absolute occupancy measurement by restriction enzyme accessibilities |
| Organisms |
Schizosaccharomyces pombe; Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
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.
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| Overall design |
calibration samples for ORE-seq: generated by mixing defined ratios of S. cerevisiae genomic DNA completely cut or uncut with a respective restriction enzyme (either AluI, BamHI or HindIII); for normalization, S. pombe genomic DNA completely digested with EcoRI was spiked into (about 10% of S. cerevisiae DNA mass) the completely cut or uncut S. cerevisiae genomic DNA prior to mixing the defined ratios.
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| Contributor(s) |
Oberbeckmannn E, Wolff MR, Krietenstein N, Schmid A, Straub T, Gerland U, Korber P |
| Citation(s) |
36807068 |
| Submission date |
Nov 18, 2021 |
| Last update date |
Apr 02, 2023 |
| Contact name |
Tobias Straub |
| E-mail(s) |
tstraub@med.uni-muenchen.de
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| Organization name |
LMU Munich
|
| Department |
Biomedical Center, Bioinformatics
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| Street address |
Großhadener Str. 9
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| City |
Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
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| Platforms (2) |
| GPL30978 |
Illumina HiSeq 1500 (Saccharomyces cerevisiae; Schizosaccharomyces pombe) |
| GPL30979 |
NextSeq 1000 (Saccharomyces cerevisiae; Schizosaccharomyces pombe) |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA781633 |
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