Method and kit for detection of protein-DNA markers, such as protein-DNA interactions and/or histone modifications, in cells
AssigneeKoninklijke Nederlandse Akademie van Wetenschappen
InventorsJop Haico Kind, Silke Johanna Anna Lochs, Robin Harm Willem Eric Van Der Weide
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Abstract
A method for sequencing DNA wherein a sample comprising isolated cell nuclei is contacted with an antibody that forms a covalent conjugate with a first DNA adapter, and wherein the formed antibody-DNA conjugate can bind a protein of interest and ligate to an end of a dephosphorylated DNA fragment, and wherein the sample is then contacted with a second DNA adapter that coheres to the first DNA adapter of the antibody-DNA conjugate to obtain a second DNA adapter—first DNA adapter—DNA fragment product that allows for sequencing of an amplified product.
Claims20
A method of detecting a target genomic DNA that binds to a polypeptide of interest among a plurality of genomic DNA, the method comprising:
- (a) providing:
- (i) a sample comprising a plurality of cells that comprise the plurality of genomic DNA, wherein the plurality of cells are permeabilized and fixated;
- (ii) an antibody-DNA adapter conjugate that comprises:
- (1) an antibody that directly or indirectly binds to the polypeptide of interest that binds to the target genomic DNA, and
- (2) a first DNA adapter that comprises a first barcode sequence, a first end, and a second end, wherein the first DNA adapter is linked to the antibody;
- (iii) a second DNA adaptor that comprises a second barcode sequence, a third end, and a fourth end;
- (b) contacting the sample with a restriction enzyme, wherein the contacting results in fragmentation of the plurality of genomic DNA into a plurality of DNA fragments that each comprises an end that is configured to bind to the first end of the first DNA adapter;
- (c) contacting the sample containing the plurality of DNA fragments with the antibody-DNA adapter conjugate, wherein the contacting results in the antibody of the antibody-DNA adaptor conjugate binding directly or indirectly to the polypeptide of interest that is bound to the target genomic DNA, thereby resulting in localization of the first DNA adaptor to the target genomic DNA;
- (d) ligating the first end of first DNA adapter to the target genomic DNA via the end produced by the restriction enzyme in (b);
- (e) ligating the third end of the second DNA adapter to the second end of the first DNA adapter via an end-to-end base pairing;
- and
- (f) sequencing the target genomic DNA ligated to the first DNA adaptor and the second DNA adaptor;
- wherein the sequencing results in detection of the target genomic DNA among the plurality of genomic DNA;
- wherein the second DNA adaptor increases detection sensitivity of the antibody-DNA adapter conjugate relative to a corresponding antibody-DNA adapter conjugate without the second DNA adaptor.
A system for detecting a target genomic DNA that binds to a polypeptide of interest among a plurality of cells that contain a plurality of nuclear targets, the system comprising:
- (a) a sample comprising the plurality of cells that are permeabilized and fixated;
- (b) a first cutting moiety that is configured to produce an end on the nuclear target upon contacting with the sample;
- (c) a binding molecule-oligo adapter conjugate that comprises a binding molecule and a first oligo adapter, wherein the binding molecule directly or indirectly binds to a polypeptide of interest that binds specifically to the nuclear target and wherein the oligo adapter comprises:
- (i) a first identifying sequence;
- (ii) a first end that is configured to bind to the end of the nuclear target from (b);
- (iii) a second end;
- (d) a joiner that joins the oligo adapter to the nuclear target;
- (e) a second oligo adapter comprising a second barcode sequence, a third end that binds to the second end of the first oligo adapter via an end-to-end base pairing, and a fourth end;
- and
- (f) reagents for amplifying and sequencing;
- wherein the second oligo adaptor increases detection sensitivity of the molecule-oligo adapter conjugate relative a corresponding molecule-oligo adapter conjugate without the second oligo adaptor.
Dependencies are inferred from the claim text.