HiC: method of identifying interactions between genomic loci
The disclosed Hi-C protocol can identify genomic loci that are spatially co-located in vivo. These spatial co-locations may include, but are not limited to, intrachromosomal interactions and/or interchromosomal interactions. Hi-C techniques may be applied to many different scales of interest. For example, on a large scale, Hi-C techniques can be used to identify long-range interactions between distant genomic loci.
1. A method for creating a ligation junction, comprising:
a) providing a cross-linked whole genome and a linker comprising a marker;
b) fragmenting said cross-linked whole genome to produce a fragmented, cross-linked whole genome, wherein said fragmented, cross-linked whole genome comprises at least one 3′ end overhang sequence and at least one 5′ end overhang sequence;
c) incorporating said linker between said at least one 3′ end overhang sequence and said 5′ end overhang sequence among a portion of the fragmented cross-linked whole genome, said incorporation creating a marked ligation junction.
2. The method of claim 1 , wherein said fragmented, cross-linked whole genome is provided from at least one fixed cell.
3. The method of claim 1 , wherein said fragmenting of step b) comprises digesting said cross-linked whole genome with at least one restriction endonuclease.
4. The method of claim 1 , further comprising digesting said fragmented, cross-linked whole genome with a restriction endonuclease wherein said 3′ end overhang sequence comprises a first sticky end and said 5′ end overhang sequence comprises a second sticky end.
5. The method of claim 1 , further comprising digesting said fragmented cross-linked whole genome with an exonuclease.
6. A method for creating a ligation junction, comprising:
a) providing a cross-linked whole genome;
b) fragmenting said cross-linked whole genome to produce a fragmented, cross-linked whole genome, wherein said fragmented, cross-linked whole genome comprises at least one 3′ end overhang sequence and at least one 5′ end overhang sequence;
c) incorporating a junction marker between said at least one 3′ end overhang sequence and at least one 5′ overhang sequence among a portion of the fragmented, cross-linked whole genome, said incorporation creating ligation junctions, said ligation junctions being marked with said junction marker.
7. The method of claim 6 , wherein said fragmented, cross-linked whole genome is provided from at least one fixed cell.
8. The method of claim 7 , wherein said at least one fixed cell comprises at least one human cell.
9. The method of claim 7 , wherein said at least one fixed cell comprises at least one non-human cell.
10. The method of claim 6 , wherein said fragmenting of step b) comprises digesting said cross-linked whole genome with at least one restriction enzyme.
11. The method of claim 6 , wherein said junction marker comprises an affinity-tag.
12. The method of claim 6 , further comprising purifying some of said ligation junctions with said junction marker.
13. The method of claim 11 , wherein said affinity tag is selected from the group consisting of a biotin tag, a polyhistidine tag, and a FLAG tag.
14. The method of claim 6 , wherein said junction marker comprises biotin.
15. The method of claim 11 , wherein said junction marker comprises a phosphodiester bond synthesized pursuant to incorporating said affinity-tagged base.
16. The method of claim 6 , wherein said junction marker comprises a phosphodiester bond.
17. The method of claim 11 , further comprising purifying some of said ligation junctions with said affinity-tag.