ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities
Contribution in Methods in Molecular Biology volume from the Korber lab, first author Elisa Oberbeckmann
21.02.2023
Elisa Oberbeckmann, Michael Roland Wolff, Nils Krietenstein, Mark Heron, Andrea Schmid, Tobias Straub, Ulrich Gerland & Philipp Korber (2023 Feb 21) ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities. In: Marinov, G.K., Greenleaf, W.J. (eds) Chromatin Accessibility. Methods in Molecular Biology, vol 2611, pp 121-152. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2899-7_9
Abstract cited directly from the volume:
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.