ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Restriction enzyme digestion is a classical method for assessing DNA accessibility in chromatin.
• This method allows for the determination of DNA accessibility or occupancy in absolute terms.

Purpose of the Study:

• To adapt the classical restriction enzyme digestion approach for genome-wide DNA accessibility analysis.
• To develop a protocol for accurate genome-wide measurement of DNA accessibility and occupancy.

Main Methods:

• Exhaustive restriction enzyme digestion of chromatin followed by DNA purification and library preparation for high-throughput sequencing.
• Bioinformatic analysis to count uncut DNA fragments and DNA ends generated by restriction enzyme digestion.
• Implementation of two orthogonal bias correction methods: "corrected cut-uncut" and "cut-all cut".

Main Results:

• The protocol enables genome-wide determination of DNA accessibility and occupancy at restriction sites.
• The developed methods correct for biases introduced during library preparation and sequencing.
• Accurate measurements of absolute DNA accessibility are achievable.

Conclusions:

• The presented protocol provides a robust method for genome-wide DNA accessibility profiling.
• This approach can be adapted for various species, starting with S. cerevisiae.
• Accurate assessment of chromatin accessibility is crucial for understanding genome regulation.