Comprehensive in vitro reporting of cleavage events by sequencing (Circle-seq)
First Claim
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1. 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.
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Abstract
Sensitive, unbiased methods for genome-wide detection of potential CRISPR-Cas9 off-target cleavage sites from cell type-specific genomic DNA samples.
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Citations
19 Claims
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1. A method of preparing a library of covalently closed circular double-stranded DNA (dsDNA) fragments, the method comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification