ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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 molecules, respectively, out of all molecules. The protocol presented here 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 uncut DNA fragments as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straightforward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments. 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 or 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.
Original language | English |
---|---|
Title of host publication | Chromatin Accessibility : Methods and Protocols |
Number of pages | 32 |
Publisher | Humana Press |
Publication date | 2023 |
Pages | 121-152 |
ISBN (Print) | 978-1-0716-2898-0, 978-1-0716-2901-7 |
ISBN (Electronic) | 978-1-0716-2899-7 |
DOIs | |
Publication status | Published - 2023 |
Series | Methods in Molecular Biology |
---|---|
Volume | 2611 |
ISSN | 1064-3745 |
Bibliographical note
Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
- Absolute occupancy, Chromatin, DNA accessibility, High-throughput sequencing, Restriction enzyme
Research areas
ID: 339139065