Methods and kits for identifying polypeptide binding sites in a genome

A method for identifying where a polypeptide of interest binds in a genome, the method comprising:

• (a) immunoprecipitating the polypeptide of interest which is cross-linked to a polynucleotide fragment using an antibody linked to a substrate;
• (b) ligating an adaptor to the polynucleotide fragment, which adaptor comprises two tail-to-tail primer sequences, a restriction site, and a nucleotide barcode, which barcode protrudes as a 5′ end overhang to prevent ligation to the barcode optionally followed by a washing step;
• (c) filling in the 5′ overhang to copy the barcode and generate blunt ends for exonuclease digestion;
• (d) digesting the blunt-ended polynucleotide fragment from step (c) with an exonuclease, which terminates digestion of the polynucleotide upon encountering a physical barrier caused by the polypeptide (stop base);
• (e) extracting the polynucleotide fragment produced by the exonuclease digestion from step (d) and purifying a single-stranded polynucleotide;
• (f) self-circularizing the single-stranded polynucleotide from step (e) to place the barcode adjacent to the stop base;
• (g) contacting the circularized polynucleotide with (1) an oligonucleotide designed to produce localized double-stranded DNA around the restriction site in the adaptor and (2) a restriction enzyme that recognizes and cleaves the circularized polynucleotide at the restriction site in the adaptor to re-linearize the polynucleotide fragment, wherein upon relinearization the polynucleotide fragment comprises a primer sequence at each end;
• and
• (h) amplifying the polynucleotide sequence to an extent sufficient for sequencing.

A kit for identifying where a polypeptide of interest binds in a genome, the kit comprising:

• (a) reagents sufficient to carry out a ChIP-exo process;
• (b) reagents sufficient to carry out a library preparation protocol adapted from an iCLIP process;
• and
• (c) instructions for use.

A kit for identifying where a polypeptide of interest binds in a genome, the kit comprising:

• (a) reagents to wash chromatin;
• (b) reagents for carrying out end repair;
• (c) reagents for carrying out dA tailing;
• (d) an adaptor;
• (e) reagents for ligating the adaptor to the chromatin;
• (f) reagents for filling in 5′ overhang in the chromatin caused by the adaptor;
• (g) reagents for end trimming;
• (h) reagents for carrying out 5′-3′ double-stranded-specific exonuclease digestion;
• (i) reagents for carrying out 5′-3′ single-stranded-specific exonuclease digestion;
• (j) reagents for carrying out self-circularization of single stranded polynucleotide sequence;
• (k) reagents for re-linearizing the circular polynucleotide sequence;
• and
• (l) reagents for carrying out PCR amplification of the re-linearized polynucleotide sequence.