Analysis of chromatin using a nicking enzyme
Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.
1. A method for analyzing open chromatin, comprising:
(a) obtaining a sample containing permeabilized cell nuclei, wherein the cell nuclei comprise chromatin;
(b) reacting the cell nuclei with a composition comprising a mixture of a nicking enzyme, four dNTPs, and at least one labeled dNTP, and a polymerase, to selectively label the chromatin; and
(c) analyzing the labeled nuclei of (b) for open chromatin, wherein the analyzing is done by:
(i) detecting an optically-detectable signal in the cell nuclei, if the at least one labeled dNTP comprises an optically detectable label; or
(ii) enriching for and then sequencing labeled DNA fragments that comprise the labeled nucleotide, if the labeled nucleotide comprises an affinity tag.
2. The method of claim 1 , wherein the at least one labeled dNTP comprises an optically detectable label and the analyzing step of (c) is performed by microscopy to produce an image of at least part of the sample.
3. The method of claim 1 , wherein the at least one labeled dNTP comprises an affinity tag and the analyzing step of (c) is performed by:
(i) fragmenting the DNA in the sample;
(ii) enriching for fragments that contain the labeled nucleotide; and
(iii) sequencing the enriched fragments.
4. The method of claim 1 , wherein the sample comprises isolated nuclei.
5. The method of claim 1 , wherein the sample comprises permeabilized cells, wherein the permeabilized cell nuclei are contained in the permeabilized cells.
6. The method of claim 5 , wherein the cells are fixed or unfixed.
7. The method of claim 1 , wherein the sample is a clinical sample.
8. The method of claim 7 , wherein the clinical sample is a tumor biopsy.
9. The method of claim 1 , wherein the nicking enzyme is methylation dependent and further comprises labeling of closed chromatin.
10. The method of claim 1 , wherein the nicking enzyme nicks at methylated CpGs.
11. The method of claim 1 , wherein the nicking enzyme is methylation insensitive, and the method further comprises labeling of open chromatin.