Methods for generating chromatin conformation capture 3C libraries

Abstract

The present invention relates to a method for generating a chromatin conformation capture (3C) library. This can be used to identify nucleic acid regions that interact with each other within a nucleic acid sample. The method comprises: treating nucleic acids in a eukaryotic cell population, the method comprising the following steps: (i) fixing nucleic acids within cells in the eukaryotic cell population; (ii) permeabilizing or removing the cell membranes of the eukaryotic cells; and (iii) fragmenting the fixed nucleic acids within the cells to produce nucleic acid fragments.

Description

Technical Field

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

[0002] Technological advances such as RNA-seq[1], ChIP-seq[2-3], DNase-seq[4], and ATAC-seq[5] have advanced our ability to annotate regulatory elements in the genome and determine their potential functions. However, an outstanding challenge is to understand the mechanisms by which regulatory elements control specific gene promoters over a distance (10s to 1000s of kb).

[0003] Using conventional chromosome conformation capture (3C), the interactions between enhancers, silencers, boundary elements, and promoters at each locus can be analyzed in detail with high resolution [6-11].

[0004] Since the development of the original 3C method in 2002 [6], several new 3C-based technologies have emerged, such as capture-C, Hi-C, capture Hi-C, in situ Hi-C, circular chromosome conformation capture (4C), 4C-seq, ChIA-PET, carbon copy chromosome conformation capture (5C), and NG capture-C [12, 13] (WO2017 / 068379). Each of these technologies has its specific advantages and disadvantages.

[0005] There remains a need for more chromosome conformation capture protocols with increased sensitivity and resolution that are easy to perform but can generate data in a high-throughput manner.

[0006] The resolution of these methods is still limited when studying mammalian genomes. In most assays, the resolution is determined by the bin size used to pool data and increase signal intensity. This is a function of sequencing depth and the size of the organism's genome (sequencing requirements increase with the square of the genome size or resolution). If sufficient sequence depth can be obtained, the limiting factor becomes the restriction fragment size, which is equal to the theoretical limit of approximately 256 bp with 4 cutting restriction enzymes (which are the highest resolution enzymes currently commonly used for 3C library preparation). Although sequence-independent nucleases such as DNaseI and micrococcal nuclease have been used previously to generate 3C libraries, the enrichment steps used in these previous protocols did not produce data with higher resolution than restriction enzymes in larger mammalian genomes [14,15].

[0007] Improved resolution may be used to highlight regulatory sequences that control genes in greater detail and to identify novel sequences that control genes. Improved resolution should also allow single nucleotide polymorphisms identified through genome-wide association studies to be associated with genes or other aspects of genome function or structure that they control with greater confidence. This has potential benefits for personalized medicine, diagnostics, and drug discovery.

[0008] The best resolution previously achievable was using Next Generation Capture-C [12,13].

[0009] The inventors have now found that a significant improvement in resolution can be obtained using the method of the present invention. Using this method, single base pair resolution can be obtained; this resolution is an order of magnitude greater than the resolution obtainable by previous methods. Summary of the invention

[0010] The methods of the present invention involve a novel combination of fixation and digestion steps in the generation of 3C libraries.

[0011] In previous methods (e.g. WO2017 / 068379), cells have been fixed (e.g. using formaldehyde) and then homogenized to break the cells and release the chromatin. In the method of the present invention, the cells are fixed and then permeabilized (to allow digestion). It has been found that this gentler method facilitates higher resolution.

[0012] Digestion of chromatin has been performed previously using a number of different enzymes including 4 and 6 base pair cutting restriction endonucleases such as HindIII, EcoRI, NcoI, XbaI, BglIII, DpnII and NlaIII, as well as bacterial nucleases including micrococcal nuclease and DNaseI.

[0013] The inventors have now found that micrococcal nuclease helps to achieve enhanced resolution when used in the method of the invention.

[0014] Although micrococcal nuclease has been used previously to map nucleosome-resolution chromosome folding in yeast [14, 15], the resolution previously achieved was reported to be between 200 bp and 4 kb. Furthermore, although yeast (Saccharomyces cerevisiae) cells are eukaryotic cells, the Saccharomyces cerevisiae genome is approximately 12 million base pairs, which is only about 1 / 250 the size of the human genome.

[0015] The methods of the invention thus allow the study of the interactions of regulatory elements in mammalian genes, inter alia, at a resolution that was previously unavailable.

[0016] In one embodiment, the present invention provides a method for processing nucleic acids in a eukaryotic cell population, the method comprising the following steps:

[0017] (i) fixing nucleic acids in cells of a eukaryotic (preferably mammalian) cell population;

[0018] (ii) permeabilizing or removing the cell membrane of said eukaryotic (preferably mammalian) cells; and

[0019] (iii) fragmenting the immobilized nucleic acid in the cell to produce nucleic acid fragments.

[0020] Preferably, the cell is a mammalian cell.

[0021] In a preferred embodiment, the present invention provides a method for processing nucleic acids in a eukaryotic (preferably mammalian) cell population, the method comprising the following steps:

[0022] (i) cross-linking nucleic acids in cells of a population of eukaryotic (preferably mammalian) cells;

[0023] (ii) permeabilizing or removing the cell membrane of said eukaryotic (preferably mammalian) cells; and

[0024] (iii) fragmenting the cross-linked nucleic acid within the cell to produce nucleic acid fragments.

[0025] Preferably, the nucleic acid is chromatin.

[0026] In another embodiment, the present invention provides a method for generating a 3C library, the method comprising the following steps:

[0027] (a) treating nucleic acid by the method for treating nucleic acid in a eukaryotic cell population of the present invention;

[0028] (b) ligating the nucleic acid fragments to produce ligated nucleic acid fragments; and

[0029] (c) de-immobilizing the ligated nucleic acid fragments (eg, uncrosslinking).

[0030] In another embodiment, the present invention provides a method for identifying nucleic acid regions that interact with each other within a nucleic acid sample, the method comprising the steps of:

[0031] Generating a 3C library by the method for generating a 3C library of the present invention;

[0032] (d) fragmenting the 3C library to generate nucleic acid fragments;

[0033] (e) optionally, adding sequencing adapters to the ends of the nucleic acid fragments and / or amplifying the nucleic acid fragments;

[0034] (f) contacting the nucleic acid fragments with a targeting nucleic acid that binds to the subpopulation of nucleic acid fragments, wherein the targeting nucleic acid is labeled with the first half of a binding pair;

[0035] (g) separating the subpopulation of nucleic acid fragments bound by the target nucleic acid using the second half of the binding pair;

[0036] (h) amplifying the isolated nucleic acid fragment subset;

[0037] (j) optionally repeating steps (f), (g) and (h) one or more times; and

[0038] (k) optionally sequencing the amplified isolated nucleic acid fragment subset.

[0039] Preferably, the targeting nucleic acid is a DNA oligonucleotide.Preferably, the nucleic acid sample is a eukaryotic cell sample, preferably a mammalian cell sample.

[0040] In another embodiment, a method for identifying allele-specific interaction spectra in SNP-containing regions of nucleic acids is provided, the method comprising the method of the present invention, the method comprising sequencing the amplified isolated nucleic acid fragment subset to identify the allele-specific interaction spectra in the SNP-containing regions.

[0041] In yet another embodiment, a kit for identifying nucleic acid regions that interact with each other in a nucleic acid sample is provided, the kit comprising a buffer and reagents for performing the method of the present invention.

[0042] In yet another embodiment, a method of identifying one or more interacting nucleic acid regions indicative of a particular disease state or condition is provided, the method comprising:

[0043] a) performing a method as defined herein on a nucleic acid sample of eukaryotic (preferably mammalian) cells obtained from a subject suffering from a particular disease state or condition;

[0044] b) quantifying the frequency of interaction between the first nucleic acid region and the second nucleic acid region; and

[0045] c) comparing the interaction frequencies in a nucleic acid sample from a subject having the disease state or condition to the interaction frequencies in a control nucleic acid sample from a healthy subject, such that differences in the interaction frequencies in the nucleic acid samples are indicative of a particular disease state or condition.

[0046] The method of the present invention relates to the processing of nucleic acids within a population of eukaryotic cells. The nucleic acids are processed in situ, i.e. within the cells.

[0047] The nucleic acid sample may comprise a population of eukaryotic cells.

[0048] Examples of eukaryotic cells include cells from animals, plants and fungi. Preferably, the eukaryotic cell is a higher eukaryotic cell or a cell from a multicellular organism. The plant may be a monocot or a dicot. In some embodiments, the eukaryotic cell is an animal cell, preferably a vertebrate cell, more preferably a mammalian cell.

[0049] Preferably, the mammalian cell is from a human, monkey, mouse, rat, rabbit, guinea pig, sheep, horse, pig, cow, goat, dog or cat. Most preferably, the mammalian cell is a human cell.

[0050] In some embodiments, the cell is an erythroid cell or a stem cell (eg, an embryonic stem cell).Preferably, the nucleic acid is obtained from a living cell.

[0051] In some preferred embodiments, the cell population consists of 10 4 Up to 10 9 cells, more preferably 10 6 Up to 10 8 In other preferred embodiments, the cell population consists of 1-10,000 cells, 10,000-1 million cells, or 1 million to 100 million cells.

[0052] As used herein, the term "nucleic acid" encompasses chromatin, DNA and RNA. Preferably, the nucleic acid is DNA or chromatin, most preferably chromatin. Chromatin comprises nucleosomes connected by internucleosomal linkers.

[0053] Step (i) comprises fixing (e.g. cross-linking) nucleic acids in cells of a eukaryotic (preferably mammalian) cell population. Fixation (e.g. cross-linking) is performed on an individual cell basis (i.e. fixation, e.g. cross-linking, in cells). Fixation (e.g. cross-linking) is performed in situ, i.e. in the nucleus. Preferably, fixation (e.g. cross-linking) is performed in substantially all or all cells of a eukaryotic (preferably mammalian) cell population.

[0054] In this step, nucleic acids (eg, within chromatin) are fixed (eg, cross-linked) so that regions within the nucleic acids that interact with each other are held or fixed in close proximity.

[0055] The nucleic acid regions that interact with each other are particularly DNA elements that influence or control the expression of related genes or other aspects of genome function or structure. For example, the DNA element can be a promoter, enhancer, insulator and / or silencer.

[0056] The nucleic acids can be immobilized by crosslinking them or in particular by embedding them in a fixative.

[0057] The nucleic acid regions that interact with each other can be crosslinked directly (i.e. nucleic acid to nucleic acid) or indirectly (e.g. by crosslinking of nucleic acid to a part that binds nucleic acid (e.g. a protein) or between proteins that bind nucleic acid directly or indirectly). Preferably, the nucleic acids are crosslinked using a crosslinking agent. The crosslinking agent must be a crosslinking agent that can enter (non-permeabilized) cells. Preferably, the crosslinking agent is formaldehyde.

[0058] Fixatives are substances that can enter (non-permeabilized) cells and fix nucleic acids in those cells so that regions of the nucleic acids that interact with each other are kept or fixed in close proximity. In some embodiments, eukaryotic (preferably mammalian) cells are fixed in a plug of fixative. Examples of fixatives include gels formed from cross-linked polymers, preferably hydrogels.

[0059] Hydrogel is a network of hydrophilic polymer chains, sometimes found as colloidal gels, wherein water is a dispersion medium. The structure of the hydrogel can be changed by changing the concentration of the polymer forming the hydrogel in the hydrogel. Examples of hydrogel polymers include polyvinyl alcohol, acrylate polymers (such as sodium acrylate) and polymers with a large amount of hydrophilic groups. Other hydrogel polymers include agarose, alginate, methylcellulose, hyaluronic acid, elastin-like polypeptides and other naturally derived polymers. Preferably, the fixing agent is agarose gel.

[0060] Step (ii) comprises permeabilizing or removing the cell membrane of the eukaryotic (preferably mammalian) cell. In this step, the outer cell membrane and nuclear membrane of the cell are at least permeabilized to allow the fragmentation enzyme to enter the nucleic acid (e.g. chromatin) in the cell nucleus.

[0061] As used herein, the term "permeabilization" means that the outer cell membrane and nuclear membrane are made permeable to fragmentation enzymes, but the membranes remain otherwise intact.

[0062] In some embodiments, the outer cell membrane and / or nuclear membrane are not lysed. In some embodiments, the outer cell membrane and / or nuclear membrane are not partially or completely destroyed. In some embodiments, the outer cell membrane and / or nuclear membrane are not partially or completely removed.

[0063] In a preferred embodiment, the outer cell membrane and nuclear membrane of the eukaryotic (preferably mammalian) cell are permeabilized without removing the outer cell membrane or nuclear membrane. In other embodiments, the cell membrane is removed and the nuclear membrane is permeabilized (but not removed). The chromatin surrounded by the permeabilized nuclear membrane can then be isolated. In other embodiments, the cell membrane is removed and the nuclear membrane is removed. The chromatin can then be isolated.

[0064] In embodiments where the cell membrane of the cells is permeabilized or removed, they are preferably permeabilized or removed in substantially all or all cells, respectively. In embodiments where the nuclear membrane of the cells is permeabilized or removed, they are preferably permeabilized or removed in substantially all or all cells, respectively.

[0065] The outer cell membrane and 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. Preferably, the permeabilizing agent is digitonin (eg from Sigma).

[0066] In one embodiment, the amount of permeabilizing agent used is an amount sufficient to permeabilize the outer cell membrane and nuclear membrane of the cell, preferably without partially or completely removing the cell membrane and / or nuclear membrane. A higher amount of permeabilizing agent can be used to completely remove the outer cell membrane and nuclear membrane of the cell. A moderate amount of permeabilizing agent can be used to completely remove the outer cell membrane and permeabilize the nuclear membrane of the cell.

[0067] Step (iii) comprises fragmenting the fixed (eg, cross-linked) nucleic acid (eg, chromatin) in the cell to produce nucleic acid fragments. The nucleic acid fragments are preferably chromatin or DNA fragments.

[0068] In this step, fixed (e.g., cross-linked) nucleic acids (e.g., chromatin) are fragmented to allow their subsequent ligation with other nucleic acid sequences within the chromatin that were in close physical proximity in the nucleus upon fixation. In this fragmentation step, internucleosomal linkers are cleaved and their length is preferably reduced.

[0069] After fragmentation, the free ends of the nucleic acids within the chromatin are ligated to each other using a ligation reaction. This generates a 3C library in which the order of the nucleic acid fragments is rearranged to reflect their proximity in 3-dimensional space at the time of fixation / immobilization, rather than their original position in the linear nucleic acid molecule.

[0070] The fragmentation step should preferably not affect the integrity of the fixation (eg, cross-linking) or not substantially affect the integrity of the fixation (eg, cross-linking).

[0071] Fragmentation can be carried out by any suitable means. Examples of fragmentation processes include the use of enzymes, such as endonucleases. In certain embodiments, restriction endonucleases are used, most preferably restriction endonucleases (such as DpnII or NlaIII) that recognize 4 base pairs are used for fragmentation.

[0072] In some embodiments of the invention, the fragmentation step does not include a step of labeling the free ends of the nucleic acid fragments with the first half of the binding pair. In particular, in some embodiments of the invention, the fragmentation step does not include a step of labeling the free ends of the nucleic acid fragments with biotin.

[0073] In a particularly preferred embodiment of the present invention, fragmentation is performed using an exonuclease. Preferably, the exonuclease is a 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 RNA, and all sequences will eventually be cut.

[0074] Fragmentation of fixed (e.g. cross-linked) nucleic acids (e.g. chromatin) is not carried out to completion. In particular, internucleosomal linkers are not all cut / digested during the fragmentation step. Preferably, fragmentation of fixed (e.g. cross-linked) nucleic acids (e.g. fixed or cross-linked chromatin) is carried out such that all or substantially all internucleosomal linkers remain intact. Preferably, chromatin is digested to produce >70% (preferably >80% or >90%) mononucleosomes.

[0075] The internucleosomal linker preferably remains at least partially intact (ie not digested to completion), but may be cleaved. Preferably, the internucleosomal linker has a length of 10-500, 10-200, 50-200 or 10-100 base pairs after fragmentation (eg digestion).

[0076] Preferably, nucleic acid (eg, chromatin) is fragmented (eg, digested) into mononucleosomes (eg, 180-200 bp), and more preferably with an internucleosomal linker attached. Preferably, nucleic acid wrapped around the histone core of the nucleosome is not fragmented (digested).

[0077] The duration of the fragmentation step and / or the amount / concentration of the fragmentation enzyme (when used) is chosen to achieve this.

[0078] Preferably, a relatively long incubation time is used in conjunction with a very small amount of enzyme. This gives greater control over the reaction than a shorter incubation time using more enzyme.

[0079] The extent of chromatin fragmentation and degradation of internucleosomal linkers can be readily determined by gel electrophoresis, for example by using an automated system such as the Agilent TapeStation (D1000 reagents).

[0080] After the fragmentation step (i.e., step (iii)), the fixed (e.g., cross-linked) nucleosomes can be linked together using DNA adapters (e.g., as shown in Ohno et al., Subnucleosomal genome structure reveals different nucleosome folding motifs, Cell (2019), https: / / doi.org / 10.1016 / j.cell.2018.12.014). For example, the DNA adapters can be connected to the DNA entry end and the DNA exit end of the DNA molecule in the nucleosome. The adapters can be labeled (e.g., biotin conjugated). In other embodiments, the adapters are not labeled (e.g., they are not biotin conjugated).

[0081] In other embodiments of the present invention, a method for generating a 3C library is provided, the method comprising the following steps:

[0082] (a) treating nucleic acid by the method of the present invention for treating nucleic acid in a eukaryotic (preferably mammalian) cell population;

[0083] (b) ligating the nucleic acid fragments to produce ligated nucleic acid fragments; and

[0084] (c) de-immobilizing the ligated nucleic acid fragments (eg, uncrosslinking).

[0085] As used herein, the term "3C library" refers to a library of DNA fragments, wherein the DNA fragments comprise consecutively linked DNA elements, wherein the DNA elements are DNA elements that are capable of interacting with each other (eg, within a cell).

[0086] Step (b) comprises ligating the nucleic acid fragments obtained from step (a) to produce ligated nucleic acid fragments. The ligated nucleic acid fragments are preferably ligated chromatin fragments or ligated DNA fragments.

[0087] In this step, the free ends of the nucleic acid fragments produced in step (iii) are ligated together to produce ligated nucleic acid fragments.

[0088] Ligation will occur between the free ends of the nucleic acid fragments in a random manner. However, ligation will most preferably occur between adjacent free nucleic acid ends that are held in close proximity to each other by the fixation (e.g., crosslinking) process of step (i). In this way, nucleic acid regions within the nucleic acid sample that previously interacted with each other will now preferably chemically connect (ligate) to each other.

[0089] Preferably, the length of the ligated nucleic acid fragments is greater than 200 bp (i.e., the fragment size in the DNA fragment length spectrum is increased, preferably such that very little DNA of that fragment size is present in the main mononucleosome peak after the digestion reaction (see Figure 2 )).

[0090] Prior to ligation, the ends of the nucleic acid fragments are preferably blunt-ended and phosphorylated, for example using T4 polynucleotide kinase (PNK) and DNA polymerase I, Large (Klenow) fragment.

[0091] Any suitable ligating agent, such as a ligase, can be used for ligation. Preferably, the ligase is a DNA ligase. Examples of suitable DNA ligases include T4 DNA ligase.

[0092] In step (c), the ligated nucleic acid fragments are de-immobilized (eg, cross-linked).

[0093] If the cells have not yet been lysed, they can be lysed at this time.

[0094] If the cell membrane has not been removed previously, the cell membrane is also removed at this time, for example, with a lysis buffer, proteinase K and heat treatment or a suitable detergent. Alternatively, a sufficient amount of a permeabilizing agent (e.g., as disclosed herein) may be used. In some preferred embodiments, the nuclear membrane and / or cell membrane is not removed until this step.

[0095] In this step, the ligated nucleic acid fragments (e.g., ligated chromatin fragments) are defixed (e.g., cross-linked) to generate linear nucleic acid fragments (e.g., linear chromatin fragments). For example, the fixative is removed / dissolved or the cross-linked portion is cut or removed.

[0096] In some embodiments, crosslinks are removed by heating the ligated nucleic acid fragments to an elevated temperature, such as 50°C, 60°C, 70°C, 80°C or higher. Proteinase K is preferably used to remove crosslinks. Optionally, non-nucleic acid substances (e.g., proteins, crosslinking agents, etc.) are also removed at this time. RNA is also preferably removed from the sample at this time, preferably using RNase. For example, the ligated nucleic acid fragments can be extracted using phenol / chloroform or solid phase extraction methods (e.g., Qiagen spin columns).

[0097] In another embodiment, the invention provides a method for identifying nucleic acid regions that interact with each other in a nucleic acid sample.

[0098] The first step of the method comprises generating a 3C library by the method of the present invention, ie, steps (a), (b) and (c) as described above.

[0099] 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 reduced to a size suitable for high-throughput sequencing, capture and / or amplification.

[0100] Preferably, the length of the nucleic acid fragments is reduced to 100-500 base pairs, more preferably 100-300 or 150-250 base pairs, and most preferably about 250 base pairs.

[0101] Fragmentation can be performed by any suitable method. Examples of suitable fragmentation methods include the use of nucleases (e.g., restriction endonucleases) and sonication. Preferably, fragmentation is performed by sonication.

[0102] In step (e), sequencing adapters are optionally added to the ends of the nucleic acid fragments. In addition, the nucleic acid fragments can be amplified at this time. In this optional step, sequencing adapters and / or amplification primers (e.g., short double-stranded nucleic acids) are added to the two ends of the nucleic acid fragments to facilitate amplification and subsequent sequencing of the nucleic acid fragments.

[0103] Each sequencing adapter can contain a unique index barcode, i.e. a short nucleic acid motif that serves as a unique identifier for the nucleic acid fragment. Preferably, the sequencing adapter is a next generation sequencing adapter. In some embodiments, the sequencing adapter comprises a P5 or P7 sequence that mediates binding to a flow cell and bridge amplification. Internal binding sites for sequencing primers and barcodes can also be added to allow the sample to be indexed. Sequencing adapters can be added to nucleic acid fragments by ligation-mediated PCR.

[0104] The nucleic acid fragments may also be amplified at this point (eg, by PCR). For example, 1-20 rounds of PCR may be performed, preferably 3-10 rounds, and most preferably about 6 rounds of PCR.

[0105] The indexed samples can now optionally be pooled for multiplex sequence analysis.

[0106] In step (f), the nucleic acid fragments are contacted with a targeting nucleic acid that binds to a subpopulation of nucleic acid fragments, wherein the targeting nucleic acid is labeled with the first half of the binding pair. In this step, the desired nucleic acid fragments (e.g., DNA fragments) are prepared for separation from the background of contaminating nucleic acid fragments.

[0107] A targeting nucleic acid having a nucleotide sequence that is complementary or substantially complementary to a nucleotide sequence of a desired region of nucleic acid in a nucleic acid sample is used. Thus, the targeting nucleic acid will hybridize to a desired region of nucleic acid in a nucleic acid sample under appropriate conditions.

[0108] For example, the desired region of nucleic acid may be a region from the promoter of a particular gene (wherein it is desired to determine which DNA regions interact with the promoter), or it may be a region of an enhancer element (wherein it is desired to determine which genes are enhanced by the element).

[0109] 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 (eg, DNA oligonucleotide).

[0110] When restriction endonucleases are used in the generation of 3C libraries, the targeting nucleic acid preferably comprises the ends of the restriction fragments containing the desired region and includes a restriction endonuclease site. In this way, the targeting nucleic acid is bound to the informative junction.

[0111] Preferably, the concentration of the targeting nucleic acid (e.g., DNA oligonucleotide) is 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 applies to each oligonucleotide used.

[0112] Preferably, the same targeting nucleic acid is used in any repetition of step (f).

[0113] Targeting nucleic acids (e.g., labeled oligonucleotides) can be designed to bind to any sequence within the genome of the organism being studied. Preferably, the targeting nucleic acid (e.g., labeled oligonucleotide) is located (i.e., designed to bind) within a nucleosome-depleted region of a promoter or regulatory element (e.g., enhancer, repressor, or CTCF binding site) of a gene or non-coding RNA of interest.

[0114] Most preferably, the targeting nucleic acid (e.g., labeled oligonucleotide) is located (i.e., designed to bind) within 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. As used herein, the term "central" region refers to the middle 50% (preferably the middle 30%, 20%, or 10%) of the sequence of the nucleosome-depleted region. The term "central" region may also refer to the middle 500, 400, 300, 200, 100, or 50 base pairs of the sequence of the nucleosome-depleted region.

[0115] In this way, a very strong and high-resolution picture of the functional interactions that control gene (or RNA) expression can be obtained. Regions of nucleosome depletion can be easily defined using assays including DNaseI hypersensitivity, ATAC-seq, and chromatin immunoprecipitation.

[0116] In some embodiments, the targeting nucleic acid is designed to bind to (or overlap with) a DNase I hypersensitive site or an ATAC sequence of a promoter of a gene or noncoding RNA of interest or a regulatory element in the nucleic acid.

[0117] In contrast, when the targeting nucleic acid (e.g., labeled oligonucleotide) is moved 1000 bp to the left or right of the central region, the physical interaction spectrum weakens and it becomes more difficult to precisely define the regulatory contacts (see Figure 4a -c).

[0118] At loci where gene regulation is well defined (e.g., the α and β globin loci, HBA and HBB), the profiles obtainable from the methods of the invention from central nucleosome-depleted regions at the promoter define all known regulatory elements down to nearly single base pair resolution (see Figure 4c ). This resolution was previously unavailable.

[0119] Transcription factor binding sites at distal regulatory elements can also be defined by signals from the central part of the promoter. This can be achieved by using the junction sites between the portion of the capture read (at the promoter) and the report read (at the enhancer). Transcription factor binding sites can be defined because there is a reduction in the density of cleavage sites at the locations where they bind to DNA. Therefore, the strongest signal appears at unprotected sites between transcription factor binding sites. This is similar to DNaseI hypersensitive footprint analysis.

[0120] Examples of binding pairs include biotin and streptavidin. Preferably, the first half of the binding pair is biotin.

[0121] In step (g), the second half of the binding pair is used to separate the subpopulation of nucleic acid fragments that have been bound by the target nucleic acid. In this step, the second half of the binding pair is combined with the first half of the binding pair. In order to help separate the target nucleic acid fragments, the second half of the binding pair can be combined with a physical support, such as a column or a bead (e.g., a magnetic bead).

[0122] For example, the first half of the binding pair can be biotin and the second half of the binding pair can be streptavidin coated beads. The target nucleic acid fragments can then be separated from the background because they will bind to the column or magnetic beads and the background nucleic acid can then be removed.

[0123] In some embodiments of the invention, the method is not performed on a microarray.

[0124] In step (h), the isolated nucleic acid fragment subset is amplified. In this step, the isolated nucleic acid fragments (e.g., DNA fragments) are amplified to enrich the desired nucleic acid fragments. Preferably, the amplification is performed by PCR. Preferably, the amplification includes 10-40 cycles of PCR amplification, more preferably 12-14 cycles.

[0125] In embodiments of the invention wherein the sequencing adaptor comprises a P5 or P7 sequence, a PCR primer that binds to the latter sequence may be used.

[0126] Steps (d)-(h) of the method of the present invention can result in an enrichment of about 5-20,000 times compared to a corresponding method without steps (f), (g) and (h).

[0127] In step (j), steps (f), (g) and (h) can be repeated (in this order). This results in greater enrichment of the desired nucleic acid fragments compared to a corresponding method without steps (f), (g) and (h), such that typically >90% of the reads contain the sequence targeted by the oligonucleotide capture. Steps (f), (g) and (h) can be repeated (in this order), for example, 1-5 times, such as 1, 2, 3, 4 or 5 times.

[0128] The steps of the method are preferably performed in the order specified.

[0129] Optionally, the method further comprises step (k), i.e. sequencing the amplified nucleic acid fragment subset. The skilled person will be fully aware of the many DNA sequencing methods that can be used. Preferably, sequencing is performed using an Illumina platform, such as Miseq, HiSeq, NextSeq or NovoSeq, using 150 bp paired end sequences (i.e. a total of 300 bp).

[0130] The methods of the present invention are performed in vitro or ex vivo.

[0131] The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety. In particular, the disclosure of WO2017 / 068379 is specifically incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 Overview of the methods of the invention for generating 3C libraries.

[0133] Figure 2 Nucleosome fragmentation profile. After DNA extraction, material was assayed using automated gel electrophoresis (Agilent TapeStation using D1000 reagents). Optimal digestion levels were obtained when chromatin was primarily digested into mononucleosomes (180-200 bp) but with internucleosomal linkers attached ( Figure 2 and 3 ). Overdigestion to <160 bp removes inter-nucleosomal linkers, meaning that it is not possible to ligate fragments in close proximity.

[0134] Figure 3Model explaining the rationale behind optimal digestion. Prior to digestion, chromatin is wrapped around nucleosomes. Approximately 148 bp are wrapped around each nucleosome, with approximately 20-80 bp of linker sequence. When samples are 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 are completely digested between nucleosomes, it is impossible to allow the ligation reaction to proceed.

[0135] Figure 4a a, b and c Comparison of data generated by different 3C methods. These figures show the increased resolution obtained using the method of the present invention compared to the data from Hsieh et al.

[15] .

[0136] Figure 4a A 100 kb portion of the alpha globin locus is shown and illustrates how small changes in the position of the oligonucleotides used for capture can dramatically alter the interaction profile. In particular, oligonucleotides placed directly at the hypersensitive site at the gene promoter show highly discrete interactions with enhancer regulatory elements that control gene expression. Data from NG capture-C and 4C-seq methods [10, 12, 13] are included to allow comparison with the best previous available methods.

[0137] Figure 4b Shows the Figure 4a The 20 kb portion of the sequence was included, and the 20 kb data from Hsieh et al.

[15] , which was generated from Saccharomyces cerevisiae, was included to allow comparison.

[0138] Figure 4c Shown from Figure 4b 1 kb portion of the enhancer region, which highlights the resolution that can be obtained from the method of the present invention. This gives close to single base pair resolution when mapping junctions, and this may highlight binding sites for transcription factors within enhancer regions.

[0139] Figure 5 Shown is a comparison of MCC performed with intact whole cell preparations versus MCC performed with nuclear preparations.

[0140] Figure 6 Micro-C data. This illustrates the nucleotide sequence resolution obtained using state-of-the-art methods (taken from Hsieh et al.

[14] ). Figure 5 ). DETAILED DESCRIPTION

[0141] Example

[0142] The present invention is further illustrated by the following examples, wherein parts and percentages are by weight unless otherwise stated, and degrees are degrees Celsius. It should be understood that these examples, although indicating preferred embodiments of the present invention, are given only by way of illustration. From the above discussion and these examples, those skilled in the art can determine the essential features of the present invention, and without departing from its spirit and scope, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, in addition to those modifications shown and described herein, from the foregoing description, various modifications of the present invention will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0143] Example 1: Preparation of Micrococcal Nuclease Chromatin Conformation Capture (MCC) Library

[0144] The method is outlined in Figure 1 Shown in.

[0145] fixed

[0146] Substitute (1-2)×10 7 The cells were fixed in 10 mL of medium with a final concentration of 2% formaldehyde at room temperature for 10 minutes. The reaction was quenched by adding 1M cold glycine (final concentration 130mM) and centrifuged at 300g / 4°C for 5 minutes. The supernatant was discarded, the cell pellet was resuspended in phosphate-buffered saline, centrifuged (300g / 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 approximately 0.05% (enough to permeabilize the cells, depending on the batch of digitonin). At this point, the cells can be quickly frozen and stored at -80°C if necessary.

[0147] Digestion

[0148] The permeabilized cells were centrifuged at 300 g for 5 min, the supernatant discarded, and the cells were resuspended in calcium-reduced micrococcal nuclease buffer (Tris HCL pH 7.5 10 mM, CaCl 2 1 mM). A titration of different concentrations of micrococcal nuclease (NEB or Worthington) was used to digest the chromatin (typically ranging from 0.5-40 Kunitz U for a reaction volume of 800 μl containing 2,000,000 cells). The reaction was incubated at 37°C on an Eppendorf Thermomixer at 800 rpm for 1 hour. The nucleosome digestion profile is shown in Figure 2 middle.

[0149] The reaction was quenched with EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (Sigma)) to a final concentration of 5 mM. 200 μl was removed as a control to measure the digestion efficiency. The reaction was centrifuged (5 minutes at 300 g) and the digestion buffer was discarded. The cells were resuspended in phosphate-buffered saline and centrifuged again (5 minutes at 300 g), and the supernatant was discarded.

[0150] connect

[0151] End repair and phosphorylation of DNA were performed before ligation. Cells were resuspended in DNA ligase buffer (Thermo Scientific, final concentration 40mM Tris HCl pH 7.5, 10mM MgCl2, 10mM DTT, 5mM ATP) supplemented with dNTPs (dATP, dCTP, dGTP and dTTP (Thermo Fischer R0191)) at a final concentration of 400 μM each) and EGTA 5mM. T4 polynucleotide kinase PNK (NEB M0201L) and DNA polymerase I (large (Klenow) fragment NEBM0210L) were added to a final concentration of 200U / ml and 100U / 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.

[0152] Decrosslinking

[0153] Chromatin was decrosslinked with proteinase K at 65°C (>2 hours) and DNA was purified using phenol chloroform with RNAse treatment (Roche: 1119915) or Qiagen DNeasy blood and tissue kit.

[0154] Assess digestion and ligation efficiency using gel electrophoresis or Agilent Tapestation (D1000 reagent). This should show >80% mononucleosomes and a significant increase in fragment size in the 3C-ligated product ( Figure 2 Over-digestion of chromatin removes inter-nucleosomal linker sequences, and when this occurs, sample ligation fails ( Figure 2 and 3 ).

[0155] Ultrasonication

[0156] The oligonucleotide capture protocol was performed as conventional next generation capture-C. Briefly, the micrococcal nuclease 3C library was sonicated to an average fragment size of 200 base pairs using a Covaris S220 focused sonicator.

[0157] Addition of sequencing adapters

[0158] Sequencing adapters were added using the NEB Ultra II kit and PCR amplification was performed using the Herculase PCR kit (Agilent). The libraries were hybridized with 120 base pair biotinylated oligonucleotides (at a concentration of 13 pM-130 fmols / sample, depending on the number of oligonucleotides used) using Roche SeqCap reagents for 72 hours.

[0159] Bead capture

[0160] Samples were captured with streptavidin beads (Thermo Fischer M270), washed and amplified using Roche SeqCap reagents and standard protocols. A second round of oligonucleotide capture was performed using the same oligonucleotides and reagents, only with a 24 hour hybridization reaction.

[0161] Sequencing

[0162] Material was sequenced using the Illumina platform with 300 base pair reads (150 base pair paired end).

[0163] result

[0164] 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 for 35 genes were generated simultaneously. The experimental design included a central capture oligonucleotide and two flanking oligonucleotides, wherein the central capture oligonucleotide was designed to capture contacts directly from the middle of the hypersensitive site at the gene promoter, one flanking oligonucleotide approximately 1 kb upstream (labeled "left") and one flanking oligonucleotide approximately 1 kb downstream (labeled "right"). The data show that the resolution of MCC is much greater than the resolution achievable by the best previously available methods for defining interaction profiles at high resolution in mammalian genomes (NG capture-C and 4C-seq). In addition, despite the much larger genome size, the data has significantly improved resolution compared to all contrast all contact maps in yeast generated using the Micro-C protocol

[14] .

[0165] The position of the oligonucleotide used for capture significantly altered the interaction profile. When the oligonucleotide was placed directly on a hypersensitive site in a gene promoter, MCC showed highly discrete interactions with enhancer regulatory elements known to control gene expression ( Figure 4a, b, c). However, when the biotinylated oligonucleotide was located approximately 1 kb upstream or downstream of the central oligonucleotide position on the DNase site, the profile changed and the interaction was more diffuse. Data from NG-capture-C and 4C-seq were included to allow comparison with the best previously available method for defining a one-versus-all interaction profile in a large mammalian genome. Figure 4b Shows the Figure 4a The 20 kb portion was also included, and the 20 kb data from Hsieh et al.

[15] generated from Saccharomyces cerevisiae was included.

[0166] Figure 4c Shown from Figure 4b This highlights the resolution obtainable from the methods of the invention. When junctions are drawn (as opposed to the pile-up of entire reads shown in other traces), this gives close to single base pair resolution. This highlights potential transcription factor binding sites within enhancer regions, similar to the DNaseI hypersensitive footprinting data. In this experiment, an additional 35 genes were analyzed, and these data show a similar improvement in resolution.

[0167] Example 2: Effect of digitonin on resolution

[0168] Figure 5 Comparison of MCC performed with intact whole cell preparations to MCC performed with nuclear preparations is shown. Whole cell preparations show much more distinct peaks with enhancer elements than data generated from nuclei. NG capture-C and 4C-seq data are included for comparison (both of which were generated from 3C libraries generated from nuclei rather than intact cells).

[0169] Comparative Example 3: Resolution obtained using the Micro-C method

[0170] For comparison purposes only, the prior art Micro-C method (Hsieh et al., 2015 and 2016 [14, 15]) is referenced. Figure 4 shows the results from Hsieh et al.

[15] (Supplementary Figure 2 ) data, showing a 20 kb region on yeast chromosome IX. Figure 6 (From Hsieh et al.

[14] Figure 5 C ) shows two 20 kb x 20 kb matrices showing wild type and ssu72-2 Micro-C data. These illustrate the lower level of resolution achieved in this Micro-C approach.

[0171] References

[0172] 1.Wang,Z.,Gerstein,M.&Snyder,M.RNA-Seq:a revolutionary tool fortranscriptomics.Nat Rev Genet 10,57-63(2009).

[0173] 2.Mikkelsen,T.S.et al.Genome-wide maps of chromatin state inpluripotent and lineage-committed cells.Nature 448,553-60(2007).

[0174] 3.Robertson,G.et al.Genome-wide profiles of STAT1 DNA associationusing chromatin immunoprecipitation and massively parallel sequencing.NatMethods 4,651-7(2007).

[0175] 4.Hesselberth,J.R.et al.Global mapping of protein-DNA interactions invivo by digital genomic footprinting.Nat Methods 6,283-9(2009).

[0176] 5.Buenrostro,J.D.,Giresi,P.G.,Zaba,L.C.,Chang,H.Y.&Greenleaf,W.J.Transposition of native chromatin for fast and sensitive epigenomicprofiling of open chromatin,DNA-binding proteins and nucleosome position.NatMethods 10,1213-8(2013).

[0177] 6.Dekker,J.,Rippe,K.,Dekker,M.&Kleckner,N.Capturing chromosomeconformation.Science 295,1306-11(2002).

[0178] 7.Tolhuis,B.,Palstra,R.J.,Splinter,E.,Grosveld,F.&de Laat,W.Loopingand interaction between hypersensitive sites in the active beta-globinlocus.Mol Cell 10,1453-65(2002).

[0179] 8.Noordermeer,D.et al.The dynamic architecture of Hox geneclusters.Science 334,222-5(2011).

[0180] 9.Sanyal,A.,Lajoie,B.R.,Jain,G.&Dekker,J.The long-range interactionlandscape of gene promoters.Nature 489,109-13(2012).

[0181] 10.van de Werken,H.J.et al.Robust 4C-seq data analysis to screen forregulatory DNA interactions.Nat Methods 9,969-72(2012).

[0182] 11.de Laat,W.&Duboule,D.Topology of mammalian developmental enhancersand their regulatory landscapes.Nature 502,499-506(2013).

[0183] 12.Davies J.O.J.,Oudelaar A.M.,Higgs D.R.and Hughes J.R.How best toidentify chromosomal interactions:a comparison of approaches.Nature Methods2017,14(2),125-134

[0184] 13.Davies J.O.J.,Telenius J.M.,McGowan S.,Roberts N.A.,Taylor S.,Higgs D.R.and Hughes J.R.‘Multiplexed analysis of chromosome conformation atvastly improved sensitivity’,Nature Methods 2016;13,74-80

[0185] 14.Hsieh T.H.,Weiner A.,Lajoie B.,Dekker J.,Friedman N.,Rando O.J.

[0186] Mapping Nucleosome Resolution Chromosome Folding in Yeast by Micro-C.Cell.2015 Jul 2;162(1):108-19.

[0187] 15.Hsieh T.S.Fudenberg G.,Goloborodko A.,Rando O.J.Micro-C XL:assaying chromosome conformation from the nucleosome to the entire genome.NatMethods.2016Dec;13(12):1009-1011。

Claims

1. A method for processing chromatin in a mammalian cell population, the method comprising: The following steps are involved: (i) cross-linking chromatin in cells of a population of mammalian cells; (ii) permeabilizing the outer cell membrane and the nuclear cell membrane of the mammalian cell to generate cross-linked chromatin within the permeabilized cell; as well as (iii) fragmenting the cross-linked chromatin within the permeabilized cells using micrococcal nuclease to generate mononucleosomes, wherein the inter-mononucleosome linkers are partially intact.

2. A method for processing nucleic acids in a mammalian cell population, the method comprising: The following steps are involved: (i) fixing nucleic acids in cells of a mammalian cell population; (ii) permeabilizing or removing the cell membrane of the mammalian cell; as well as (iii) fragmenting the immobilized nucleic acid in the cell using micrococcal nuclease to generate mononucleosomes, wherein the inter-mononucleosome linker is partially intact.

3. The method of claim 2, wherein the nucleic acid is chromatin comprising nucleosomes connected by mononucleosomal inter-nucleosomal linkers.

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

5. The method of claim 2, wherein in step (ii): (a) the outer cell membrane and / or nuclear membrane is not lysed; or (b) the outer cell membrane or nuclear membrane has not been partially or completely removed; or (c) the outer cell membrane and nuclear membrane of the cell are permeabilized but not removed; or (d) the cell membrane is removed and the nuclear membrane is permeabilized but not removed; or (e) The cell membrane is removed and the nuclear membrane is removed.

6. The method of claim 2, wherein in step (iii), the immobilized nucleic acid is fragmented so as to generate mononucleosomes, wherein the inter-mononucleosome linker is partially intact.

7. The method of claim 2, wherein the number of cells in the mammalian cell population is 1-10,000.

8. The method of claim 2, wherein the number of cells in the mammalian cell population is 10,000-1,000,000.

9. The method of claim 2, wherein the number of cells in the mammalian cell population is between 1 million and 100 million.

10. A method for generating a chromatin conformation capture (3C) library, the method The following steps are involved: (a) treating the nucleic acid by a method as defined in any one of claims 2 to 9; (b) ligating the nucleic acid fragments to produce ligated nucleic acid fragments; and (c) deimmobilizing the ligated nucleic acid fragments.

11. A method for generating a chromatin conformation capture (3C) library, the method The following steps are involved: (a) treating chromatin by a method as defined in claim 1; and ligating the nucleic acid fragments to produce ligated nucleic acid fragments; as well as (b) decrosslinking the ligated nucleic acid fragments.

12. A method for identifying nucleic acid regions that interact with each other within a nucleic acid sample, the method comprising: The following steps are involved: Producing a 3C library by the method as defined in claim 10 or 11; (c) fragmenting the 3C library to generate nucleic acid fragments; (d) contacting the nucleic acid fragments with a targeting nucleic acid that binds to the subpopulation of nucleic acid fragments, wherein the targeting nucleic acid is labeled with the first half of a binding pair; (e) separating the subpopulation of nucleic acid fragments bound by the target nucleic acid using the second half of the binding pair; as well as (f) amplifying the isolated nucleic acid fragment subset; To identify nucleic acid regions that interact with each other within the nucleic acid sample.

13. The method of claim 12, wherein the method further comprises include: adding sequencing adapters to the ends of the nucleic acid fragments and / or amplifying the nucleic acid fragments; Repeating steps (d), (e) and (f) one or more times; and / or The amplified, isolated subset of nucleic acid fragments is sequenced.

14. The method of claim 12, wherein the targeting nucleic acid is a DNA oligonucleotide. The method of claim 12 , wherein the concentration of the target nucleic acid is 5 μM to 1 pM.

16. The method of claim 12, wherein the concentration of the targeting nucleic acid is 2.9 μM to 29 pM. The method of claim 12 , wherein the concentration of the target nucleic acid is 1 μM to 30 pM.

18. The method of claim 12, wherein the concentration of the target nucleic acid is 300 nM to 30 pM.

19. The method of claim 12, wherein the targeting nucleic acid is selected to bind within a nucleosome-depleted region of a promoter or regulatory element of a gene or non-coding RNA of interest in the nucleic acid.

20. The method of claim 19, wherein the targeting nucleic acid is selected to bind within the central region of a nucleosome-depleted region of a promoter or regulatory element of a gene or non-coding RNA of interest in the nucleic acid.

21. The method of claim 19, wherein the regulatory element is an enhancer, a repressor, or a CTCF binding site.

22. The method of claim 12, wherein the targeting nucleic acid is designed to bind to or overlap with a DNaseI hypersensitive site or an ATAC sequence of a promoter of a gene or non-coding RNA of interest or a regulatory element in a nucleic acid.

23. The method of claim 12, wherein steps (d), (e), and (f) are repeated once.

24. The method of claim 12, wherein steps (d), (e), and (f) are repeated twice.

25. The method of claim 12, wherein steps (d), (e), and (f) are repeated three times.

26. The method of claim 12, wherein steps (d), (e), and (f) are repeated 4 times.

27. The method of claim 12, wherein steps (d), (e), and (f) are repeated 5 times.

28. A method for identifying allele-specific interaction spectra in SNP-containing regions of nucleic acids, the method comprising the method of claim 12, the method comprising sequencing the amplified isolated nucleic acid fragment subset to identify the allele-specific interaction spectra in the SNP-containing regions.

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