Comprehensive in vitro reporting of cleavage events by sequencing (Circle-seq)

A method of preparing a library of covalently closed circular double-stranded DNA (dsDNA) fragments, the method comprising:

• providing a sample comprising dsDNA;
• randomly shearing the dsDNA to a defined average length to provide a population of dsDNA fragments;
• preparing the fragments for end-ligation;
• ligating to the ends of the fragments a stem-loop adapter comprising a single deoxyuridine adjacent to or within a single-stranded loop sequence comprising a palindromic sequence for intramolecular ligation, to prepare a population of ligated linear dsDNA fragments;
• contacting the population of ligated linear dsDNA fragments with an exonuclease to degrade any remaining linear fragments with unligated ends, to produce a purified population of ligated linear dsDNA fragments,
• contacting the purified population of ligated linear dsDNA fragments with enzymes that nick the ligated dsDNA fragments at the deoxyuridine and remove a 3′ terminal phosphate;
• incubating the nicked linear dsDNA fragments under conditions sufficient to promote intramolecular ligation and formation of circular dsDNA molecules;
• purifying the ligated circular dsDNA fragments by using an exonuclease to degrade any unligated non-circular fragments, thereby preparing a library of covalently closed fully circular dsDNA fragments.