Methods for identifying macromolecule interactions

1. A method for identifying interactions of DNA, RNA, and/or protein molecules in a cell, comprising:

(i) lysing the cell to form a cell lysate comprising DNA, RNA, and/or protein molecules;

(ii) distributing the DNA, RNA, and/or protein molecules into a plurality of first suspensions;

(iii) adding ligation adaptor molecules to each of the first suspensions, the ligation adaptor molecules configured to modify at least one end of each of the DNA, RNA, and/or protein molecules in the first suspensions and capable of ligating to a unique first nucleotide tag;

(iv) adding the unique first nucleotide tag to each of the first suspensions to tag the DNA, RNA and/or protein molecules in the respective first suspension, thereby forming a plurality of tagged first suspensions;

(v) pooling the plurality of tagged first suspensions to form a first tagged pool;

(vi) distributing the DNA, RNA, and/or protein molecules of the first tagged pool into a plurality of second suspensions;

(vii) adding a unique second nucleotide tag to each of the plurality of second suspensions to tag the DNA, RNA, and/or protein molecules in the respective second suspension, thereby forming a plurality of tagged second suspensions;

(viii) pooling the plurality of tagged second suspensions to form a second tagged pool comprising the DNA, RNA, and/or protein molecules;

repeating distributing the DNA, RNA, and/or protein molecules and steps (iv)-(viii) at least once, whereby the DNA, RNA, and/or protein molecules each comprises a set of unique nucleotide tags;

obtaining sequence information for the DNA, RNA, and/or protein molecules and their respective set of unique nucleotide tags; and

identifying the DNA, RNA, and/or protein molecules tagged with the same set of unique nucleotide tags as likely interacting molecules;

wherein the ligation adaptor molecules configured to modify at least one end of the DNA molecules comprise a 3′ spacer that allows the nucleotide tags to only ligate to the 5′ end of each single-stranded DNA sequence and prevents formation of hairpins during the step of obtaining sequence information.

2. The method of claim 1 , further comprising diluting the cell lysate prior to distributing the cell lysate into the plurality of first suspensions.

3. The method of claim 1 , further comprising:

adding an antibody modified with an oligonucleotide to the cell lysate.

4. The method of claim 1 , further comprising adding a crosslinker to the cell prior to lysing the cell or after lysing the cell.

5. The method of claim 1 , further comprising shearing chromatin in the cell lysate.

6. A method for identifying interactions of DNA, RNA, and/or protein molecules in a cell, comprising:

(i) lysing the cell to form a cell lysate;

(ii) distributing the cell lysate into a plurality of lysate suspensions;

(iii) adding ligation adaptor molecules to each of the lysate suspensions, the ligation adaptor molecules configured to modify at least one end of each of the DNA, RNA, and/or protein molecules and capable of ligating to a unique nucleotide tag;

(iv) adding the unique nucleotide tag to each of the lysate suspensions to tag the DNA, RNA, and/or protein molecules in the respective lysate suspension and thereby forming a plurality of tagged lysate suspensions, the unique nucleotide tag in each tagged lysate suspension being different from the unique nucleotide tags for the other tagged lysate suspensions;

(v) pooling the plurality of tagged lysate suspensions to form a tagged pool;

(vi) repeating distributing the tagged pool into a plurality of tagged suspensions and performing (iv) and (v) on the plurality of tagged suspensions, wherein the repeating is performed n number of times to form a final tagged pool comprising a plurality of tagged suspensions in which the DNA, RNA, and/or protein molecules have n+1 number of unique nucleotide tags;

(vii) obtaining sequence information for the DNA, RNA, and/or protein molecules and their n+1 number of nucleotide tags; and

(viii) identifying the DNA, RNA, and/or protein molecules having the same sequence and order of nucleotide tags as likely interacting molecules;

wherein the ligation adaptor molecules configured to modify at least one end of the DNA molecules comprises a 3′ spacer that allows the nucleotide tags to only ligate to the 5′ end of each single-stranded DNA sequence and prevents formation of hairpins during step (vii).

7. A method for detecting interactions of molecules in a nucleus of a cell, comprising:

(i) lysing the cell;

(ii) isolating the nucleus from the cell lysate;

(iii) shearing the chromatin in the nucleus forming a suspension of sheared chromatin;

(iv) distributing the suspension into a plurality of suspensions;

(v) modifying at least one end of each of the DNA, RNA, and/or protein molecules in the suspension of sheared chromatin with ligation adaptor molecules, the ligation adaptor molecules capable of ligating to a unique nucleotide tag;

(vi) adding the unique nucleotide tag to DNA, RNA, and/or protein molecules of the sheared chromatin in each of the plurality of suspensions, each unique nucleotide tag being different for each suspension, thereby forming a plurality of tagged suspensions;

(vii) pooling the plurality of tagged suspensions to form a tagged pool;

repeating distributing the suspension of sheared chromatin and steps (vi)-(vii) at least once, whereby the DNA, RNA, and/or protein molecules each comprise a set of unique nucleotide tags;

obtaining sequence information for the DNA, RNA, and/or protein molecules of the sheared chromatin and their respective set of unique nucleotide tags; and

identifying the DNA, RNA, and/or protein molecules having the same set of unique nucleotide tags as likely interacting molecules;

wherein the ligation adaptor molecules that modifies at least one end of the DNA molecules comprises a 3′ spacer that allows the nucleotide tags to only ligate to the 5′ end of each single-stranded DNA sequence and prevents formation of hairpins during the step of obtaining sequence information.

8. The method of claim 7 , further comprising:

adding an antibody modified with an oligonucleotide to the cell prior to isolating the nucleus.

9. The method of claim 7 , further comprising adding a crosslinker to the cell prior to lysing the cell or after lysing the cell.

10. The method of claim 1 , wherein the unique nucleotide tags anneal with a 7 nucleotide overhang.

11. The method of claim 6 , wherein the unique nucleotide tags anneal with a 7 nucleotide overhang.

12. The method of claim 7 , wherein the unique nucleotide tags anneal with a 7 nucleotide overhang.