Process for producing a chromatin conformation capture (3C) library
The present invention relates to a process for producing a chromatin conformation capture (3C) library. This may be used for identifying nucleic acid regions within a nucleic acid sample which interact with one another. The process comprises treating nucleic acids in a population of eukaryotic cells, the process comprising the steps: (i) immobilising the nucleic acids within the cells in a population of eukaryotic cells; (ii) permeabilising or removing the cell membranes of the eukaryotic cells; and (iii) fragmenting the immobilised nucleic acids within the cells to produce nucleic acid fragments.
1 . A method comprising producing nucleic acids with unlabeled ends, comprising:
(a) treating chromatin in a population of eukaryotic cells by:
(i) immobilising the chromatin within the cells in the population of eukaryotic cells;
(ii) permeabilising or removing cell membranes of the eukaryotic cells; and
(iii) fragmenting the immobilised chromatin within the cells to produce nucleic acids with unlabeled ends comprising >70% mono-nucleosomes of 180-200 bp;
(b) ligating the nucleic acids with unlabeled ends comprising the mono-nucleosomes of 180-200 bp to produce ligated nucleic acid fragments; and
(c) de-immobilising the ligated nucleic acid fragments.
2 . The method of claim 1 , wherein in Step (i), the chromatin is immobilised by cross-linking the chromatin.
3 . The method of claim 1 , wherein:
(a) the outer cell membranes and nuclear membranes are permeabilised;
(b) the outer cell membranes and/or nuclear membranes are not lysed;
(c) the outer cell membranes or nuclear membranes are not completely removed;
(d) the outer cell membranes and nuclear membranes of the cells are permeabilised without removing the outer cell membranes or nuclear membranes;
(e) the cell membranes are removed and the nuclear membranes are permeabilised but not removed;
(f) the cell membranes are removed and the nuclear membranes are removed; or
(g) the process comprises (ii) permeabilising the cell membranes of the eukaryotic cells; and (iii) fragmenting the immobilised chromatin within the cells to produce nucleic acid fragments.
4 . The method of claim 1 , wherein in Step (iii), the immobilised chromatin is fragmented using an endo-exonuclease or using micrococcal nuclease.
5 . The method of claim 1 , wherein in Step (iii), the immobilised chromatin is fragmented such that mono-nucleosomes are produced, wherein the inter-nucleosomal linkers are not digested to completion.
6 . The method of claim 1 , wherein the eukaryotic cells are mammalian cells and wherein the number of cells in the population of mammalian cells is 1-10,000, 10,000-1 million, or 1 million to 100 million.
7 . A kit for identifying nucleic acid regions within a nucleic acid sample which interact with one another, the kit comprising buffers and reagents for performing a method as defined in claim 1 .
8 . The method of claim 1 , wherein the eukaryotic cells are mammalian cells.
9 . The method of claim 1 , wherein the fragmenting in step (iii) is carried out using micrococcal nuclease.
10 . The method of claim 1 , wherein the fragmenting is performed without use of a restriction endonuclease.
11 . The method of claim 1 , wherein the fragments comprise mono-nucleosomes and linkers that are not digested to completion.
12 . The method of claim 6 , wherein the mammal is a human.