Process for producing chromatin conformation capture (3C) library

Abstract

To provide a process for producing a chromatin conformation capture (3C) library, which may be used for identifying nucleic acid regions within a nucleic acid sample which interact with one another.SOLUTION: The process comprises treating nucleic acids in a population of eukaryotic cells, the process comprising the steps of: (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.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a process for generating chromatin conformation capture (3C) libraries, which can be used to identify nucleic acid regions that interact with each other within a nucleic acid sample.

[0002] Technological advances such as RNA-seq analysis [Non-Patent Document 1], ChIP-seq analysis [Non-Patent Documents 2-3], DNase-seq analysis [Non-Patent Document 4], and ATAC-seq analysis [Non-Patent Document 5] are improving our ability to annotate regulatory elements in genomes and identify their potential functions. However, elucidating the mechanisms by which regulatory elements control specific gene promoters from distant locations (10s-1000s kb) remains an unsolved challenge.

[0003] Using conventional chromosome conformation capture (3C) methods, it is possible to perform detailed, high-resolution analysis of the interactions between enhancers, silencers, boundary elements, and promoters at individual gene loci [Non-patent literature 6-11].

[0004] Since the development of the first 3C method in 2002 [Non-Patent Document 6], several new techniques based on the 3C method have been developed, such as Capture-C, Hi-C, Capture Hi-C, in situ Hi-C, Circularized Chromosome Conformation Capture (4C), 4C-seq, ChIA-PET, Carbon Copy Chromosome Conformation Capture (5C), and NG Capture-C [Non-Patent Documents 12, 13] (Patent Document 1). Each of these techniques has its own strengths and weaknesses.

[0005] There remains a need for more chromosome conformation capture protocols with improved sensitivity and resolution that are simple to implement yet capable of generating high-throughput data.

[0006] The resolution of these methods remains insufficient when studying mammalian genomes. In most assays, the resolution is determined by the bin size used to accumulate data and improve signal intensity. It is a function of the sequencing depth and the size of the organism's genome (sequencing requirements increase proportionally to the square of the genome size or resolution). If it is possible to obtain sufficient sequencing depth, the limiting factor is the size of the restriction fragment, which corresponds to a theoretical limit of 256 bp using four-cutter restriction enzymes (the highest resolution enzymes currently commonly used in 3C library preparation). Sequence-independent nucleases such as deoxyribonuclease I and micrococcal nuclease have been used to generate 3C libraries, but the enrichment steps used in these conventional protocols have not yielded data with a resolution higher than that of restriction enzymes in larger mammalian genomes [14, 15].

[0007] The increased resolution may help to highlight the regulatory sequences that control genes in greater detail and identify novel sequences that control genes. It may also allow single nucleotide polymorphisms identified by genome-wide association studies to be linked with greater confidence to the genes they control or to other aspects of genome function or structure, which may be beneficial for personalized medicine, diagnostics, and drug discovery.

[0008] The highest resolution achieved so far was obtained with the next-generation Capture-C method [12, 13].

[0009] The inventors have found that using the process of the present invention, resolution can be significantly improved: using this process, single base pair resolution can be achieved, which is more than an order of magnitude greater than is possible with conventional methods.

[0010] The process of the present invention involves a novel combination of fixation and digestion steps in the generation of a 3C library.

[0011] In conventional methods (e.g., US Patent No. 5,999,336), cells are fixed (e.g., using formaldehyde) and then homogenized to break down the cells and release the chromatin. In the process of the present invention, the cells are fixed, but then permeabilized (to allow digestion). This gentler approach has been found to contribute to higher resolution.

[0012] Digestion of chromatin has been previously performed using a number of different enzymes, including four- and six-base pair cutting restriction endonucleases such as HindIII, EcoRI, NcoI, XbaI, BglIII, DpnII, and NlaIII, and bacterial nucleases including micrococcal nuclease and deoxyribonuclease I. The inventors have found that micrococcal nuclease contributes to the increased resolution achieved when used in the process of the invention.

[0013] Micrococcal nuclease has been used previously to map nucleosome-resolution chromosome folding in yeast [14, 15], but it has been argued that the resolution achieved so far has been between 200 bp and 4 kb. Furthermore, although yeast (S. cerevisiae) cells are eukaryotic, the S. cerevisiae genome is approximately 12 million base pairs, which is only about 1 / 250 the size of the human genome.

[0014] The process of the invention therefore makes it possible to study the interactions of regulatory elements, particularly in mammalian genes, with a resolution not previously available.

[0015] In one embodiment, the invention is a process for processing nucleic acids in a eukaryotic cell population, comprising: (i) in a population of eukaryotic (preferably mammalian) cells, immobilizing nucleic acid within said cells; (ii) permeabilizing or removing the cell membrane of said eukaryotic (preferably mammalian) cells; and (iii) fragmenting the immobilized nucleic acid in the cell to produce nucleic acid fragments. The cells are preferably mammalian cells.

[0016] In a preferred embodiment, the invention provides a process for nucleic acid processing in a eukaryotic, preferably mammalian, cell population comprising the steps of: (i) in a population of eukaryotic (preferably mammalian) cells, crosslinking nucleic acids within said cells; (ii) permeabilizing or removing the cell membrane of said eukaryotic (preferably mammalian) cells; and (iii) fragmenting the crosslinked nucleic acid in the cell to produce nucleic acid fragments. The nucleic acid is preferably chromatin.

[0017] In another embodiment, the present invention provides (a) processing nucleic acid according to a process of the invention for processing nucleic acid in a eukaryotic cell population; (b) ligating the nucleic acid fragments to produce a ligated nucleic acid fragment; and (c) de-immobilizing (e.g., de-crosslinking) the ligated nucleic acid fragments.

[0018] In another embodiment, the present invention is a method for identifying nucleic acid regions that interact with each other in a nucleic acid sample, comprising the steps of: generating a 3C library by a process of the invention for generating a 3C library; (d) fragmenting the 3C library to generate nucleic acid fragments; (e) an optional step of adding sequencing adaptors to the ends of the nucleic acid fragments and / or amplifying the nucleic acid fragments; (f) contacting the nucleic acid fragments with a targeting nucleic acid that binds to a subgroup of the nucleic acid fragments, the targeting nucleic acid being labeled with the first half of a binding pair; (g) isolating the subgroup of nucleic acid fragments to which the targeting nucleic acid is bound using the second half of the binding pair; (h) amplifying a subgroup of the isolated nucleic acid fragments; (j) optionally repeating steps (f), (g), and (h) one or more times; and (k) the optional step of sequencing an isolated subgroup of said amplified nucleic acid fragments.

[0019] The targeting nucleic acid is preferably a DNA oligonucleotide.The nucleic acid sample is a eukaryotic cell sample, preferably a mammalian cell sample.

[0020] In yet another embodiment, there is provided a method for identifying an allele-specific interaction profile in a SNP-containing region of a nucleic acid, comprising the method of the invention comprising sequencing an isolated subgroup of said amplified nucleic acid fragments to identify an allele-specific interaction profile in the SNP-containing region.

[0021] In yet another embodiment, a kit for identifying nucleic acid regions that interact with each other in a nucleic acid sample is provided that contains buffers and reagents for carrying out the methods of the invention.

[0022] In yet another embodiment, a method for identifying one or more interacting nucleic acid regions indicative of a particular disease state or disorder, comprising: a) performing the methods described herein on a nucleic acid sample of eukaryotic (preferably mammalian) cells obtained from a subject having a particular disease state or disorder; b) quantifying the frequency of interactions between the first nucleic acid region and the second nucleic acid region; and c) comparing the frequency of interactions in said nucleic acid sample from a subject having said disease state or disorder with the frequency of interactions in a control nucleic acid sample from a healthy subject, wherein a difference in the frequency of interactions between said nucleic acid samples is indicative of a particular disease state or disorder.

[0023] The process of the invention relates to the processing of nucleic acids in a eukaryotic cell population. The nucleic acids are processed in situ, i.e. intracellularly. The nucleic acid sample may comprise a eukaryotic cell population.

[0024] Examples of eukaryotic cells include animal, plant and fungal cells. Preferably, said eukaryotic cell is a higher eukaryotic cell or a cell of a multicellular organism.

[0025] The plant may be a monocotyledonous or dicotyledonous plant. In some embodiments, the eukaryotic cell is an animal cell, preferably a vertebrate cell, and more preferably a mammalian cell.

[0026] Preferably, the mammalian cells are human, monkey, mouse, rat, rabbit, guinea pig, sheep, horse, pig, cow, goat, dog or cat cells, and most preferably, the mammalian cells are human cells.

[0027] In some embodiments, the cells are red blood cells or stem cells (eg, embryonic stem cells).Preferably, the nucleic acid is obtained from a living cell.

[0028] In some preferred embodiments, the cell population is 4 ~10 9 It consists of 10 cells 6 ~10 8 In another preferred embodiment, the cell population consists of 1 to 10,000 cells, 10,000 to 1 million cells, or 1 million to 100 million cells.

[0029] The term "nucleic acid" as used herein encompasses chromatin, DNA, and RNA. The nucleic acid is preferably DNA or chromatin, and most preferably chromatin. Chromatin comprises nucleosomes linked by internucleosomal linkers.

[0030] Step (i) comprises, in a population of eukaryotic (preferably mammalian) cells, immobilizing (e.g. crosslinking) nucleic acids within said cells. The immobilization (e.g. crosslinking) is performed on an individual cell basis (i.e. intracellular immobilization, e.g. crosslinking). The immobilization (e.g. crosslinking) is performed in situ, i.e. within the cell nuclei. Preferably, the immobilization (e.g. crosslinking) is performed within substantially all or all cells in a population of eukaryotic (preferably mammalian) cells.

[0031] In this process, the nucleic acid (eg, within chromatin) is immobilized (eg, cross-linked) such that regions within the nucleic acid that have interacted with each other are held or fixed in close proximity.

[0032] The nucleic acid regions that interact with each other are in particular DNA elements that affect or control the expression of related genes or other aspects of genome function or structure. For example, said DNA elements may be promoters, enhancers, insulators, and / or silencers.

[0033] The nucleic acids may be immobilized by cross-linking the nucleic acids or by embedding the nucleic acids in a particular immobilizing agent.

[0034] Regions of nucleic acids that have interacted with each other may be crosslinked directly (i.e., between nucleic acids) or indirectly (e.g., by crosslinking nucleic acids to sites (e.g., proteins) that bind to the nucleic acid, or by crosslinking proteins that are directly or indirectly bound to the nucleic acid). Nucleic acids are preferably crosslinked using a crosslinking reagent. The crosslinking agent must be capable of entering (non-permeabilized) cells. The crosslinking agent is preferably formaldehyde.

[0035] The fixation agent is a substance that can enter a (non-permeabilized) cell and immobilize the nucleic acid within the cell such that regions within the nucleic acid that have interacted with each other are held in close proximity or fixed. In some embodiments, eukaryotic (preferably mammalian) cells are immobilized within a plug of the fixation agent. Examples of fixation agents include gels, preferably hydrogels, formed from cross-linked polymers.

[0036] A hydrogel is a network of hydrophilic polymer chains and may be a colloidal gel with water as the dispersion medium. The structure of the hydrogel can be altered by changing the concentration of the hydrogel-forming polymer in the hydrogel. Examples of hydrogel polymers include polyvinyl alcohol, acrylate polymers (e.g., sodium acrylate), and polymers rich in hydrophilic groups. Other hydrogel polymers include agarose, alginate, methylcellulose, hyaluronan, elastin-like polypeptides, and other naturally occurring polymers. The fixative is preferably an agarose gel.

[0037] Step (ii) involves permeabilizing or removing the cell membrane of a eukaryotic (preferably mammalian) cell, which at least permeabilizes the outer cell membrane and the nuclear membrane of said cell in order to allow the fragmentation enzyme access to the nucleic acids (e.g., chromatin) within the nucleus.

[0038] As used herein, the term "permeabilize" means that the outer cell membrane and nuclear membrane are treated to make them permeable to a fragmentation enzyme, but that these membranes remain otherwise intact.

[0039] In some embodiments, the outer cell membrane and / or the nuclear membrane are not lysed.

[0040] In some embodiments, the outer cell membrane and / or the nuclear membrane are not partially or completely disrupted. In some embodiments, the outer cell membrane and / or the nuclear membrane are not partially or completely removed.

[0041] In a preferred embodiment, the outer cell membrane and nuclear membrane of a eukaryotic (preferably mammalian) cell are permeabilized without removing the outer cell membrane or the nuclear membrane. In another embodiment, the cell membrane is removed and the nuclear membrane is permeabilized (but not removed). The chromatin enclosed in the permeabilized nuclear membrane may then be isolated. In another embodiment, the cell membrane is removed and the nuclear membrane is removed. The chromatin may then be isolated.

[0042] In embodiments in which the plasma membrane of the cells is permeabilized or removed, it is preferred that the plasma membrane is permeabilized or removed, respectively, in substantially all or all of the cells. In embodiments in which the nuclear membrane of the cells is permeabilized or removed, it is preferred that the nuclear membrane is permeabilized or removed, respectively, in substantially all or all of the cells.

[0043] The outer cell membrane and the nuclear membrane are permeabilized using a membrane permeabilizing agent. Examples of membrane permeabilizing agents include digitonin, saponin, tergitol type NP40, Triton X-100, sodium dodecyl sulfate, and Tween 20. The permeabilizing agent is preferably digitonin (e.g., Sigma).

[0044] In one embodiment, the amount of permeabilizing agent used is sufficient to permeabilize the outer plasma membrane and nuclear membrane of the cells, preferably without partially or completely removing said plasma membrane and / or nuclear membrane. Higher amounts of permeabilizing agent may be used to completely remove the outer plasma membrane and nuclear membrane of the cells. Intermediate amounts of permeabilizing agent may be used to completely remove the outer plasma membrane and permeabilize the nuclear membrane.

[0045] Step (iii) comprises fragmenting the fixed (e.g. cross-linked) nucleic acid (e.g. chromatin) in the cell to generate nucleic acid fragments, preferably chromatin or DNA fragments.

[0046] In this process, the fixed (e.g., cross-linked) nucleic acids (e.g., chromatin) are fragmented so that they can subsequently ligate to other nucleic acid sequences within the chromatin that were in physical proximity in the nucleus at the time of fixation. In this fragmentation process, internucleosomal linkers are cleaved and preferably shortened in length.

[0047] Following fragmentation, the free ends of the nucleic acids within the chromatin are joined together using a ligation reaction, resulting in a 3C library in which the order of the nucleic acid fragments is rearranged to reflect their proximity in three-dimensional space at the time of immobilization / fixation, rather than their original location within a linear nucleic acid molecule.

[0048] Preferably, the fragmentation step does not affect or does not substantially affect the integrity of the immobilization (eg, cross-linking).

[0049] Fragmentation can be carried out by any suitable method. An example of fragmentation process includes the use of enzymes, such as endonucleases. In some embodiments, fragmentation is carried out by using restriction endonucleases, most preferably by using the restriction endonucleases that recognize 4 base pairs (e.g., DpnII or NlaIII).

[0050] In some embodiments of the invention, the fragmentation step does not include labeling the free ends of the nucleic acid fragments with a first partner of a binding pair, particularly in some embodiments of the invention, the fragmentation step does not include labeling the free ends of the nucleic acid fragments with biotin.

[0051] In a particularly preferred embodiment of the invention, fragmentation is performed using an endo-exonuclease, preferably micrococcal nuclease (EC 3.1.31.1). Micrococcal nuclease preferentially digests single-stranded nucleic acids. The enzyme is also active against double-stranded DNA and double-stranded RNA, eventually cleaving all sequences.

[0052] Fragmentation of immobilized (e.g., cross-linked) nucleic acid (e.g., chromatin) is not carried out to completion. In particular, the internucleosomal linkers are not cleaved / digested in their entirety during the fragmentation step. Fragmentation of immobilized (e.g., cross-linked) nucleic acid (e.g., immobilized or cross-linked chromatin) is preferably carried out such that all or substantially all of the internucleosomal linkers remain intact. The chromatin is preferably digested such that more than 70% (preferably more than 80% or more than 90%) mononucleosomes are produced.

[0053] The internucleosomal linker is preferably kept at least partially intact (i.e., not completely digested), but may be cleaved. The internucleosomal linker is preferably 10-500, 10-200, 50-200, or 10-100 base pairs in length after fragmentation (e.g., digestion).

[0054] The nucleic acid (e.g., chromatin) is preferably fragmented (e.g., digested) into mononucleosomes (e.g., 180 to 200 bp), and more preferably has an internucleosomal linker added thereto. The nucleic acid wrapped around the histone core of the nucleosome is preferably not fragmented (digested).

[0055] The duration of the fragmentation step and / or the amount / concentration of fragmentation enzyme (if used) are selected to achieve this.

[0056] It is preferred to use relatively long incubation times in combination with very small amounts of enzyme, which allows for greater control of the reaction compared to shorter incubation times and higher amounts of enzyme.

[0057] The degree of chromatin fragmentation and the extent of degradation of internucleosomal linkers can be easily analyzed by gel electrophoresis, for example by using an automated system such as the Agilent TapeStation (D1000 Reagents).

[0058] After the fragmentation step (i.e., step (iii)), the immobilized (e.g., cross-linked) nucleosomes may be linked together using DNA adaptors (e.g., as shown in Ohno et al., Sub-nucleosomal Genome Structure Reveals Distinct Nucleosome Folding Motifs, Cell (2019), https: / / doi.org / 10.1016 / j.cell.2018.12.014). For example, the DNA adaptors may be ligated to the DNA entry and exit ends of the DNA molecules within the nucleosome. The adaptors may be labeled (e.g., biotin-conjugated). In other embodiments, the adaptors are unlabeled (e.g., not biotin-conjugated).

[0059] In another embodiment of the present invention, (a) processing nucleic acids according to a process of the invention for processing nucleic acids in a eukaryotic (preferably mammalian) cell population; (b) ligating the nucleic acid fragments to produce a ligated nucleic acid fragment; and (c) de-immobilizing (e.g., de-crosslinking) the ligated nucleic acid fragments.

[0060] As used herein, the term "3C library" refers to a library of DNA fragments, the DNA fragments comprising contiguously linked DNA elements that are capable of interacting with each other (e.g., within a cell).

[0061] Step (b) comprises ligating the nucleic fragments obtained in step (a) to produce ligated nucleic acid fragments, preferably ligated chromatin fragments or ligated DNA fragments.

[0062] In this step, the free ends of the nucleic acid fragments produced in step (iii) are ligated to produce ligated nucleic acid fragments.

[0063] Ligation occurs randomly between the free ends of the nucleic acid fragments. However, it is most preferred that ligation occurs between adjacent free nucleic acid ends that are held in close proximity to each other by the immobilization (e.g., cross-linking) process of step (i). In this way, nucleic acid regions within the nucleic acid sample that previously interacted with each other are now preferably chemically bound (ligated) to each other.

[0064] The length of the ligated nucleic acid fragments is preferably 200 bp or greater (i.e., there is an increase in fragment size in the DNA fragment length profile, and preferably there is little fragment-sized DNA in the main mononucleosome peak after the digestion reaction) (see Figure 2).

[0065] Prior to ligation, the ends of the nucleic acid fragments are preferably blunt-ended and phosphorylated using, for example, T4 polynucleotide kinase (PNK) and DNA polymerase I, Large (Klenow) Fragment.

[0066] Ligation may be performed using any suitable ligation agent, such as a ligase. Preferably, the ligase is a DNA ligase. An example of a suitable DNA ligase is T4 DNA ligase.

[0067] In step (c), the ligated nucleic acid fragments are de-immobilized (e.g., de-crosslinked). If the cells have not already been lysed, they may be lysed at this time.

[0068] If the cell membrane has not been removed earlier, it is also removed at this time, for example using a lysis buffer, proteinase K, and heat treatment or a suitable detergent. Alternatively, a sufficient amount of a permeabilizing agent (such as those disclosed herein) may be used. In some preferred embodiments, the nuclear membrane and / or cell membrane is not removed until this step.

[0069] In this step, the ligated nucleic acid fragments (e.g., ligated chromatin fragments) are de-immobilized (e.g., de-crosslinked) to generate linear nucleic acid fragments (e.g., linear chromatin fragments), e.g., by removing / dissolving the immobilizing agent or cleaving or removing crosslinked sites.

[0070] In some embodiments, crosslinks are removed by heating the ligated nucleic acid fragments to 50° C., 60° C., 70° C., 80° C., or higher. Decrosslinking is preferably accomplished using proteinase K. Removal of non-nucleic acid material (e.g., proteins, crosslinkers, etc.) is also optionally accomplished at this time. It is also preferred to remove RNA from the sample at this point, preferably using ribonuclease. For example, the ligated nucleic acid fragments may be extracted with phenol / chloroform or solid phase extraction methods (e.g., Qiagen spin columns).

[0071] In yet another embodiment, the present invention provides a method for identifying nucleic acid regions that interact with each other in a nucleic acid sample.

[0072] The first step of the method is to generate a 3C library by the process of the present invention, ie, comprising steps (a), (b) and (c) described above.

[0073] In step (d), the nucleic acid fragments in the 3C library are fragmented. The nucleic acid fragments are preferably DNA fragments. In this step, the length of the nucleic acid fragments in the 3C library is preferably shortened to a size suitable for high-throughput sequencing, capture, and / or amplification.

[0074] Preferably, the length of the nucleic acid fragment is shortened to 100 to 500 base pairs, more preferably 100 to 300 or 150 to 250 base pairs, and most preferably about 250 base pairs.

[0075] Fragmentation may be carried out by any suitable process. Examples of suitable fragmentation processes include the use of nucleases (e.g., restriction endonucleases) and sonication. Preferably, fragmentation is carried out by sonication.

[0076] Step (e) optionally includes the addition of sequencing adaptors to the ends of the nucleic acid fragments. Additionally, the nucleic acid fragments may be amplified at this time. In this optional step, sequencing adaptors and / or amplification primers (e.g., short double-stranded nucleic acids) are added to both ends of the nucleic acid fragments to facilitate amplification and subsequent sequencing of the nucleic acid fragments.

[0077] Each sequencing adapter may contain a unique index barcode, i.e., a short nucleic acid motif that serves as a unique identifier for that nucleic acid fragment. The sequencing adapter is preferably a next-generation sequencing adapter. In some embodiments, the sequencing adapter contains a P5 or P7 sequence that mediates binding to a flow cell and bridge amplification. Internal binding sites for sequencing primers and barcodes may be added to allow sample indexing. The sequencing adapter may be added to the nucleic acid fragment by ligation-mediated PCR.

[0078] The nucleic acid fragment may be amplified at this time (for example, by PCR). For example, 1 to 20 rounds of PCR may be performed, preferably 3 to 10 rounds, and most preferably about 6 rounds of PCR.

[0079] The indexed samples may now be optionally pooled for multiplex sequence analysis.

[0080] In step (f), the nucleic acid fragments are contacted with a targeting nucleic acid that binds to a subgroup of the nucleic acid fragments, where the targeting nucleic acid is labeled with a first partner of a binding pair. This step provides for isolation of the desired nucleic acid fragments (e.g., DNA fragments) from a background of contaminating nucleic acid fragments.

[0081] A target nucleic acid is used that has a nucleotide sequence that is complementary or substantially complementary to the nucleotide sequence of a desired region of a nucleic acid in a nucleic acid sample, and thus hybridizes, under appropriate conditions, to the desired region of the nucleic acid in the nucleic acid sample.

[0082] For example, the desired region of the nucleic acid may be a region of a promoter from a particular gene (where it is desired to determine which DNA region interacts with that promoter) or a region of an enhancer element (where it is desired to determine which gene is enhanced by that element).

[0083] The targeting nucleic acid may be single-stranded or double-stranded, preferably single-stranded. The targeting nucleic acid may be DNA or RNA, preferably DNA (e.g., a DNA oligonucleotide).

[0084] When using restriction endonucleases to generate a 3C library, the targeting nucleic acid preferably comprises the end of the restriction fragment that contains the desired region and contains a restriction endonuclease site, such that the targeting nucleic acid is bound to an informative ligation junction.

[0085] The concentration of the targeting nucleic acid (e.g., DNA oligonucleotide) is preferably 5 μM to 1 pM. More preferably, the concentration of the targeting nucleic acid (e.g., DNA oligonucleotide) is 2.9 μM to 29 pM. Even more preferably, the concentration of the targeting nucleic acid (e.g., DNA oligonucleotide) is 1 μM to 30 pM, or 300 nM to 30 pM. Even more preferably, the concentration of the targeting nucleic acid (e.g., DNA oligonucleotide) is 30 nM to 0.3 nM. Most preferably, the concentration of the targeting nucleic acid (e.g., DNA oligonucleotide) is about 2.9 nM. This is true for each oligonucleotide used.

[0086] When step (f) is repeated, it is preferred to use the same targeting nucleic acid.

[0087] The targeting nucleic acid (e.g., a labeled oligonucleotide) can be designed to bind to any sequence in the genome of the organism under study. The targeting nucleic acid (e.g., a labeled oligonucleotide) is preferably located (i.e., designed to bind) within the nucleosome-depleted region of the promoter or regulatory element (e.g., enhancer, repressor, or CTCF binding site) of the gene or non-coding RNA of interest.

[0088] The targeting nucleic acid (e.g., labeled oligonucleotide) is most preferably located within (i.e., designed to bind to) the central region of the nucleosome-depleted region of the promoter or regulatory element (e.g., enhancer, repressor, or CTCF binding site) of the gene or non-coding RNA of interest. The term "central" region as used herein refers to the central 50% (preferably the central 30%, 20%, or 10%) of the sequence of the nucleosome-depleted region. The term "central" region may also refer to the central 500, 400, 300, 200, 100, or 50 base pairs of the sequence of the nucleosome-depleted region.

[0089] In this way, a very powerful and high-resolution picture of the functional interactions that control gene (or RNA) expression can be obtained. The nucleosome-depleted regions can be easily defined using assays such as DNaseI hypersensitivity, ATAC-seq, and chromatin immunoprecipitation.

[0090] In some embodiments, the targeting nucleic acid is designed to bind to (or overlap) a DNase I hypersensitive region or an ATAC sequence in the promoter of a gene or non-coding RNA of interest, or a regulatory element in the nucleic acid.

[0091] In contrast, moving the targeting nucleic acid (e.g., a labeled oligonucleotide) 1000 bp to the left or right of the central region attenuates the physical interaction profile and makes it more difficult to precisely define regulatory contacts (see Figures 4a-c).

[0092] For loci where gene regulation has already been clearly defined (e.g., alpha globin, beta globin, HBA, and HBB), profiles obtained by the method of the present invention from the central nucleosome-depleted regions of the promoters define all of the known regulatory elements at nearly single base pair resolution (see FIG. 4c), a resolution that has not previously been achieved.

[0093] Transcription factor binding sites of distal regulatory elements can also be defined from signals from the middle of the promoter. This can be achieved by using junctions between part of the capture read (at the promoter) and the reporter read (at the enhancer). The transcription factor binding sites can be defined because where they bind to the DNA there is a reduction in cut-site density. Thus, the strongest signals occur at unprotected sites between the transcription factor binding sites. This is similar to DNaseI hypersensitivity foot printing assays.

[0094] An example of a binding pair is the pair biotin and streptavidin. Preferably, the first partner of the binding pair is biotin.

[0095] In step (g), the second partner of the binding pair is used to isolate the subgroup of nucleic acid fragments to which the targeting nucleic acid is bound. In this step, the second partner of the binding pair is allowed to bind to the first partner of the binding pair. To aid in the isolation of the targeted nucleic acid fragments, the second partner of the binding pair may be bound to a physical support, such as a column or beads (e.g., magnetic beads).

[0096] For example, the first partner of the binding pair may be biotin and the second partner may be a streptavidin coated bead. The targeted nucleic acid fragments may then be isolated from the background by taking advantage of the fact that they bind to columns or magnetic beads, and the background nucleic acids may then be removed.

[0097] In some embodiments of the invention, the method is not performed on a microarray.

[0098] In step (h), a subgroup of the isolated nucleic acid fragments is amplified. In this step, the isolated nucleic acid fragments (e.g., DNA fragments) are amplified to enrich for the desired nucleic acid fragment. The amplification is preferably carried out by PCR. Preferably, the amplification comprises 10 to 40 cycles, more preferably 12 to 14 cycles of PCR amplification.

[0099] In embodiments of the invention in which the sequencing adapter comprises a P5 or P7 sequence, a PCR primer that binds to the latter sequence may be used.

[0100] Steps (d) through (h) of the method of the present invention may achieve approximately a 5-fold to 20,000-fold enrichment compared to a corresponding method that does not perform steps (f), (g), and (h).

[0101] In step (j), steps (f), (g) and (h) may be repeated (in this order). This improves the enrichment of the desired nucleic acid fragments compared to the corresponding method without steps (f), (g) and (h), and more than 90% of the reads frequently contain sequences that are targets of oligonucleotide capture. Steps (f), (g) and (h) may be repeated (in this order), for example, 1 to 5 times, for example, 1, 2, 3, 4 or 5 times.

[0102] The method steps are preferably performed in the order specified.

[0103] The method further comprises an optional step (k), i.e. sequencing the amplified subgroup of nucleic acid fragments. Those skilled in the art will be familiar with the numerous DNA sequencing methods that can be used. The sequencing is preferably performed using an Illumina platform, e.g. Miseq, HiSeq, NextSeq or NovoSeq, with 150bp paired-end sequences (i.e. 300bp in total).

[0104] The methods of the present invention may be carried out in vitro or ex vivo.

[0105] The disclosures of each document described herein are specifically incorporated herein by reference in their entirety. In particular, the disclosure of U.S. Patent No. 5,399,633 is specifically incorporated herein by reference. [Brief description of the drawings]

[0106] [Figure 1] Overview of the method of the present invention for generating 3C libraries [Diagram 2] Nucleosome fragmentation profile. After DNA extraction, the material was assayed using automated gel electrophoresis (Agilent TapeStation, D1000 reagents). The optimal level of digestion was obtained when the majority of chromatin was digested to mononucleosomes (180-200 bp) with the addition of internucleosomal linkers (Figures 2 and 3). Overdigestion below 160 bp removed the internucleosomal linkers, which meant that ligation of adjacent fragments was not possible. [Diagram 3] A model explaining the rationale for optimal digestion. Before digestion, chromatin wraps around nucleosomes. Approximately 148 bp wraps around each nucleosome, with a linker sequence of around 20-80 bp. When the sample is digested to a peak fragment size of 180-200 base pairs, the linkers between nucleosomes are cleaved but not digested. This allows ligation reactions to proceed between different nucleosomes. If the linkers between nucleosomes are completely digested, it is not possible for the ligation reaction to proceed. [Figure 4a]Comparison of data generated by different 3C methods. These panels show the higher resolution obtained using our method compared to the data of Hsieh et al.

[15] . Figure 4a shows a 100 kb section of the alpha globin locus, showing that small changes in the position of the oligonucleotides used for capture dramatically change the interaction profile. In particular, oligonucleotides placed directly across hypersensitive sites in the gene's promoter show highly individual interactions with enhancer regulatory elements that control gene expression. To allow comparison with the best methods available to date, Figure 4a also includes data from NG Capture-C and 4C-seq [10, 12, 13]. [Figure 4b] FIG. 4b shows a 20 kb section extracted from FIG. 4a and also includes for comparison 20 kb of data extracted from Hsieh et al.

[15] generated from S. cerevisiae. [Figure 4c] Figure 4c shows a 1 kb section extracted from Figure 4b, highlighting the resolution of the data obtained by the method of the invention. Plotting the ligation junctions provides near single base pair resolution and may highlight transcription factor binding sites within enhancer regions. [Diagram 5] FIG. 5 shows a comparison of MCC performed with intact whole cell preparations and with nuclear preparations. [Figure 6] Micro-C data showing the nucleotide sequence resolution obtained using prior art methods (taken from Figure 5 of Hsieh et al.

[14] ). EXAMPLES

[0107] The present invention is further illustrated by the following examples. In the following examples, unless otherwise specified, parts and percentages are parts and percentages by weight, and temperatures are in degrees Celsius. These examples are intended to illustrate preferred embodiments of the present invention and are to be construed as being for illustrative purposes only. From the above description and these examples, one skilled in the art can grasp the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope thereof. Thus, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0108] Example 1: Preparation of a micrococcal nuclease chromatin conformation capture (MCC) library An overview of this method is shown in Figure 1.

[0109] (Fixed) 1~2×10 7 The cells were fixed in 10 ml of medium containing 2% final formaldehyde for 10 min at room temperature. The reaction was quenched by adding 1 M cold glycine (130 mM final concentration) and centrifuged at 300 g for 5 min at 4° C. The supernatant was discarded and the cell pellet was resuspended in phosphate buffered saline, centrifuged (300 g, 4° C.) and the supernatant was discarded. The cell pellet was then resuspended in phosphate buffered saline and digitonin (Sigma) was added to a final concentration of up to 0.05% (sufficient to permeabilize the cells, depending on the batch of digitonin). If necessary, cells can be flash frozen at this point and stored at −80° C.

[0110] (digestion) Permeabilized cells were centrifuged at 300 g for 5 min, the supernatant discarded, and the cells resuspended in reduced calcium content micrococcal nuclease buffer (Tris HCL pH 7.5 10 mM; CaCl2 1 mM). Different concentrations of titrated micrococcal nuclease (NEB or Worthington) were used to digest chromatin (usually ranging from 0.5 to 40 Kunitz U for a reaction volume of 800 μl containing 2,000,000 cells). The reaction was incubated for 1 h at 37 °C in an Eppendorf Thermomixer at 800 rpm. The nucleosome digestion profile is shown in Figure 2. The reaction was quenched with EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (Sigma)) at a final concentration of 5 mM. 200 μl was removed as a control to measure digestion efficiency. The reaction was centrifuged (300 g for 5 min) and the digestion buffer was discarded. The cells were resuspended in phosphate buffered saline, centrifuged again (300 g for 5 min) and the supernatant was discarded.

[0111] (Ligation) DNA end-repair and phosphorylation were performed prior to ligation. Cells were resuspended in DNA ligase buffer (Thermo Scientific; final concentrations of 40 mM Tris HCl pH 7.5, 10 mM MgCl2, 10 mM DTT, 5 mM ATP) supplemented with dNTPs (dATP, dCTP, dGTP, and dTTP (Thermo Fischer, R0191), each at a final concentration of 400 uM) and 5 mM EGTA. T4 polynucleotide kinase PNK (NEB M0201L) and DNA polymerase I (Large (Klenow) Fragment NEB M0210L) were added to final concentrations of 200 U / ml and 100 U / ml, respectively, and the reaction was incubated at 37°C for 1 hour. T4 DNA ligase (Thermo Scientific, High Concentration Ligase (30 U / μl) EL0013) was added to a final concentration of 300 U / ml and the reaction was incubated overnight at 16° C. using an Eppendorf Thermomixer at 800 rpm.

[0112] (decrosslinking) Chromatin was decrosslinked with proteinase K at 65°C (>2 h) and DNA was purified using either RNAse-treated phenol-chloroform (Roche: 1119915) or Qiagen's DNeasy blood and tissue kit. Digestion and ligation efficiency was assessed using either gel electrophoresis or the Agilent Tapestation (D1000 Reagents). This should show >80% mononucleosomes and a significant increase in fragment size of the 3C ligation product (Figure 2). Overdigestion of chromatin will remove linker sequences between nucleosomes and the sample will not be able to ligate (Figure 2 and Figure 3).

[0113] (Ultrasonic Treatment) The oligonucleotide capture protocol was the same as that used in the conventional Next Generation Capture-C method. Briefly, the micrococcal nuclease 3C library was sonicated using a Covaris S220 Focused Ultrasonicator to an average fragment size of 200 base pairs.

[0114] (Adding Sequencing Adapters) Sequencing adapters were added using the NEB Ultra II kit, and PCR amplification was performed using the Herculase PCR kit (Agilent). These libraries were conventionally hybridized with 120 base pair biotinylated oligonucleotides (concentrations of 13 pm to 130 fmols / sample, depending on the number of oligonucleotides used) using Roche SeqCap reagent for 72 hours.

[0115] (Capture by beads) The samples were captured with streptavidin beads (Thermo Fischer M270), washed, and amplified using the Roche SeqCap reagents and standard protocols. A second round of oligonucleotide capture was performed using the same oligonucleotides and reagents, with only a 24-hour hybridization reaction.

[0116] (Sequencing) The material was sequenced using an Illumina platform with 300 base pair reads (150 base pair paired end).

[0117] (result) The data were analyzed as shown in Figure 4. Figure 4 shows data from a Micrococcal nuclease Capture-C (MCC) experiment. In this experiment, data were generated simultaneously for 35 genes. The experimental design included a central capture oligonucleotide designed to capture direct contacts from the center of the hypersensitive site in the gene's promoter, and two flanking oligonucleotides, one up to 1 kb upstream (labeled "left") and one up to 1 kb downstream (labeled "right"). The data show that the resolution of MCC is much higher than that achievable with the best methods available so far for defining high-resolution interaction profiles in mammalian genomes (NG Capture-C and 4C-seq). Moreover, when compared to yeast all v all contact maps generated using the Micro-C protocol

[14] , the data show substantially improved resolution, despite the much larger genome size.

[0118] The position of the oligonucleotide used for capture dramatically changes the interaction profile. When the oligonucleotide is placed directly across a hypersensitive site in the promoter of a gene, MCC reveals highly specific interactions with enhancer regulatory elements known to control gene expression (Figure 4a, 4b, 4c). However, placing biotinylated oligonucleotides approximately 1 kb upstream or downstream from the central oligo position at the DNase site changes the profile, resulting in more diffuse interactions. The NG Capture-C and 4C-seq data are presented together to allow comparison with the best available method to date for defining one vs all interaction profiles for large mammalian genomes. Figure 4b shows a 20 kb section extracted from Figure 4a and includes 20 kb data extracted from Hsieh et al.

[15] generated from S. cerevisiae.

[0119] Figure 4c shows a 1 kb section extracted from Figure 4b, highlighting the resolution obtained by the method of the present invention. Plotting the ligation junctions (as opposed to the pile-up of all reads shown in the other tracks) gives a resolution approaching single base pair resolution. Similar to the DNase I hypersensitive footprinting data, it highlights potential transcription factor binding sites within enhancer regions. Another 35 genes were analyzed in this experiment, and these data show a similar increase in resolution.

[0120] Example 2: Effect of digitonin on resolution Figure 5 shows a comparison of MCC performed with intact whole cell preparations and nuclear preparations. The whole cell preparations show much clearer peaks of enhancer elements compared to data generated from nuclei. NG Capture-C and 4C-seq data are shown together for comparison (both data were generated from 3C libraries made from nuclei but not from intact cells).

[0121] [Comparative Example 3: Resolution Obtained by Micro-C Method] For comparison purposes only, reference is made to the prior art Micro-C method (Hsieh et al., 2015 & 2016 [Non-Patent Documents 14, 15]). Figure 4 shows data extracted from Hsieh et al. [Non-Patent Document 15] (Supplementary Figure 2) showing a 20 kb region of yeast chromosome 9, and Figure 6 herein (reproduced from Figure 5C of Hsieh et al. [Non-Patent Document 14]) shows two 20 kb x 20 kb matrices showing wild type and ssu72-2 Micro-C data, illustrating the lower level of resolution achieved with the Micro-C method. [Prior art documents] [Patent documents]

[0122] [Patent Document 1] International Publication No. 2017 / 068379 Brochure [Non-patent literature]

[0123] [Non-Patent Document 1] Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10, 57-63 (2009). [Non-Patent Document 2] Mikkelsen, TS et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553-60 (2007). [Non-Patent Document 3] Robertson, G. et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods 4, 651-7 (2007). [Non-Patent Document 4] Hesselberth, JR et al. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat Methods 6, 283-9 (2009). [Non-Patent Document 5] Buenrostro, JD, Giresi, PG, Zaba, LC, Chang, HY & Greenleaf, WJ Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10, 1213-8 (2013).

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Claims

1. 1. A process for processing chromatin in a mammalian cell population, comprising: (i) in a population of mammalian cells, crosslinking chromatin within said cells; (ii) permeabilizing the outer and nuclear cell membranes of said mammalian cells; and (iii) fragmenting the crosslinked chromatin in the permeabilized cells using micrococcal nuclease to generate mononucleosomes, wherein the internucleosomal linkers are at least partially intact.

2. 1. A process for processing nucleic acids in a eukaryotic cell population, comprising: (i) in a population of eukaryotic cells, immobilizing nucleic acid within said cells; (ii) permeabilizing or removing the cell membrane of the eukaryotic cell; and (iii) fragmenting the immobilized nucleic acid in the cell to produce nucleic acid fragments; Preferably, said eukaryotic cell is a mammalian cell.

3. The process of claim 2 , wherein the nucleic acid is chromatin comprising nucleosomes linked by internucleosomal linkers.

4. 4. The process of claim 2 or claim 3, wherein in step (i) the nucleic acid is immobilized by cross-linking the nucleic acid.

5. 5. The process according to claim 2, wherein in step (ii) the outer cell membrane and the nuclear membrane are permeabilized.

6. The process according to any one of claims 2 to 5, wherein in step (iii) the immobilized nucleic acid is fragmented using an endo-exonuclease, preferably using micrococcal nuclease.

7. 7. The process of any one of claims 2 to 6, wherein in step (iii) the immobilized nucleic acid is fragmented such that mononucleosomes are generated and the internucleosomal linkers are at least partially intact.

8. 8. The process of any one of claims 2 to 7, wherein the number of cells in the mammalian cell population is between 1 and 10,000, between 10,000 and 1 million, or between 1 million and 100 million.

9. (a) processing nucleic acids by a process according to any one of claims 2 to 8, (b) ligating the nucleic acid fragments to produce a ligated nucleic acid fragment; and (c) removing the immobilization (preferably decrosslinking) of the ligated nucleic acid fragments.

10. 1. A method for identifying nucleic acid regions that interact with each other in a nucleic acid sample, comprising: Producing a 3C library by the process of claim 9, (d) fragmenting the 3C library to generate nucleic acid fragments; (e) optionally adding sequencing adaptors to the ends of the nucleic acid fragments and / or amplifying the nucleic acid fragments; (f) contacting the nucleic acid fragments with a targeting nucleic acid that binds to a subgroup of the nucleic acid fragments, the targeting nucleic acid being labeled with the first half of a binding pair; (g) isolating the subgroup of nucleic acid fragments to which the targeting nucleic acid is bound using the second half of the binding pair; (h) amplifying a subgroup of the isolated nucleic acid fragments; (j) optionally repeating steps (f), (g), and (h) one or more times; and (k) optionally sequencing an isolated subgroup of said amplified nucleic acid fragments to identify regions of nucleic acid that interact with each other within said nucleic acid sample.

11. The method of claim 10, wherein the targeting nucleic acid is a DNA oligonucleotide.

12. The method according to claim 10 or claim 11, wherein the concentration of said targeting nucleic acid (preferably a DNA oligonucleotide) is between 5 μM and 1 pM, preferably between 2.9 μM and 29 pM, more preferably between 1 μM and 30 pM or between 300 nM and 30 pM.

13. The method of claim 11 or 12, wherein the targeting nucleic acid (preferably a DNA oligonucleotide) is selected to be capable of binding within a nucleosome-depleted region of a promoter of a gene or non-coding RNA of interest, or within a nucleosome-depleted region of a regulatory element (preferably an enhancer, repressor, or CTCF binding site) in the nucleic acid.

14. The method of claim 13, wherein the targeting nucleic acid (preferably a DNA oligonucleotide) is selected capable of binding within the central region of a nucleosome-depleted region of a promoter of a gene or non-coding RNA of interest or within the central region of a nucleosome-depleted region of a regulatory element (preferably an enhancer, repressor or CTCF binding site) in said nucleic acid.

15. 15. The method of any one of claims 10 to 14, wherein step (j) is repeated 1, 2, 3, 4, or 5 times.

16. 16. A method for identifying an allele-specific interaction profile in a SNP-containing region of a nucleic acid, comprising sequencing an isolated subgroup of the amplified nucleic acid fragments to identify an allele-specific interaction profile in the SNP-containing region, comprising the method of any one of claims 10 to 15.

17. 1. A method for identifying one or more interacting nucleic acid regions that are indicative of a particular disease state or disorder, comprising: a) carrying out the method of any one of claims 10 to 15 on a nucleic acid sample of mammalian cells obtained from a subject having a particular disease state or disorder; b) quantifying the frequency of interactions between the first nucleic acid region and the second nucleic acid region; and c) comparing the frequency of interactions in said nucleic acid samples obtained from subjects having said disease state or disorder with the frequency of interactions in control nucleic acid samples obtained from healthy subjects, wherein a difference in the frequency of interactions between said nucleic acid samples is indicative of a particular disease state or disorder.

18. A kit for identifying nucleic acid regions that interact with each other in a nucleic acid sample, comprising buffers and reagents for carrying out the method according to any one of claims 1 to 17.

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