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 - 42 . (canceled)
43 . A method for detecting open chromatin in fixed or unfixed cell nuclei, comprising:
(a) reacting a sample containing fixed or unfixed cell nuclei, wherein the cell nuclei comprise chromatin, with:
(i) a reaction mixture comprising a nicking enzyme, and
(ii) a ligase and adaptors; and
(b) enriching for, optionally amplifying, and then sequencing DNA fragments that comprise the adaptors.
44 . The method of claim 43 , wherein the nicking enzyme is methylation-dependent, methylation-sensitive, or methylation-insensitive.
45 . The method of claim 43 , wherein the nicking enzyme is Nt.CviPII.
46 . The method of claim 43 , wherein the reaction mixture further comprises a polymerase, four dNTPs, and a labeled dNTP.
47 . The method of claim 46 wherein, after reaction step (a), the method comprises reverse cross-linking and isolating labelled genomic DNA, and then fragmenting the isolated labelled genomic DNA, prior to reaction step (b); optionally wherein the reverse cross-linking is done using heat or chemical treatment; optionally wherein the DNA is fragmented to a median size in the range of 100bp-lkg.
48 . The method of claim 47 , further comprising performing end-repair and/or dA tailing, and then adaptor ligation.
49 . The method of claim 43 , wherein the adaptors comprise an affinity tag.
50 . The method of claim 46 , wherein the labelled dNTP is affinity labeled.
51 . The method of claim 50 , wherein step (b) comprises capturing affinity-labelled DNA fragments using a capture moiety; optionally wherein the capture moiety is linked to a solid phase.
52 . The method of claim 51 , wherein the affinity label is biotin, and the capture moiety comprises streptavidin.
53 . The method of claim 43 , wherein step (b) comprises amplifying; optionally using barcoded primers and/or wherein the adaptors comprise a barcode sequence.
54 . The method of claim 43 , further comprising mapping a sequence read obtained from the sequencing of step (b) onto a genomic sequence.
55 . The method of claim 43 , further comprising: analyzing a first population of cells using the method claim 43 to produce a first data set and optionally a first epigenetic map; and
analyzing a second population of cells using the same method to produce a second data set and optionally a second epigenetic map; and comparing the first data set or epigenetic map to the second data set or epigenetic map.
56 . The method of claim 55 , wherein the first population of cells and the second population of cells are from the same individual at different times.
57 . The method of claim 55 , wherein the first population of cells and the second population of cells are different populations of cells from different tissues or from different individuals.
58 . The method of claim 43 , wherein the cell nuclei are obtained from a cell culture.
59 . The method of claim 43 , wherein the cell nuclei are obtained from a sample from an individual, optionally selected from a bodily fluid and a biopsy.