A method of profiling covalent DNA modification at a cellular or nuclei level and a kit for use in the method

Abstract

The present invention relates to a method of profiling covalent DNA modification(s) at a cellular or nuclei level comprising the steps of: providing a sample solution, comprising cell(s) comprising a nucleus comprising genomic DNA; adding a buffer comprising a nucleosome depletion agent to expose the genomic DNA; adding a fusion protein comprising a DNA modification binding domain and a nuclease part; incubating the fusion protein together with the exposed genomic DNA under conditions allowing the fusion protein to bind to the genomic DNA; washing the solution of step (d) with buffer to remove any excess fusion protein; incubating the solution of step (e) under conditions allowing the fusion protein to cut the genomic DNA in proximity of the DNA modification(s); and determining the sequence of the fusion protein incubated DNA of step (e) by high-throughput sequencing, thereby identifying the position(s) of the DNA modification(s) of the genomic DNA that have been sequenced. In other aspects, the invention also relates to a kit for use in performing the method.

Description

[0001] A method of profiling covalent DNA modifica^on at a cellular or nuclei level and a kit for use in the method Technicalfield The present inven^on refers to thefields of epigene^cs, genomics, DNA methyla^on profiling and single-cell and spa^al omics. More specifically, the present inven^on refers to a method of profiling covalent DNA modifica^on at a cellular or nuclei level and a kit for use in the method. Background DNA methyla^on, also known as 5-methylcytosine (5mC), represents a cri^cal form of covalent DNA modifica^on. DNA methyla^on regulates gene ac^vity and expression within a DNA segment without 10 altering the sequence itself. Deposi^on and removal of 5mC is crucial for normal development in mammals and plays a pivotal role in several fundamental biological processes, including aging and the development of cancer. Analysis of DNA methyla^on profiles in the genome is cri^cal for understanding its func^on. Several methods based on 1) bisulfite conversion (e.g., Frommer et al.,Proc Natl Acad Sci U S A.1992 Mar 1;89(5): 1827-31), 2) affinity enrichment (Taiwo et al., NatureProtocols 7, 617-636 (2012)) or 3) restric^on diges^on have been developed for this purpose. Thereare also approaches based on enzyma^c conversion (Vaisvila et al., Genome Res.2021 Jul; 31(7): 1280-1289) or long-read sequencing (Liu et al., Genome Biology 22, 295 (2021)). Whole-genome bisulfite sequencing (WGBS) is considered the gold standard for comprehensive DNA methyla^on profiling. During WGBS, genomic DNA undergoes treatment with sodium bisulfite, which specifically20 converts unmethylated cytosine (C) into uracil (U), while maintaining methylated cytosine residuesunchanged, thereby allowing for a detailed analysis of methyla^on pa^erns across the genome. When profiling genome-wide DNA methyla^on, WGBS has to generate a vast amount of data toobtain high quality DNA methyla^on profiles, leading to high cost of sequencing for each sample (>15,000 kr) and problems with data storage and handling. WGBS does not scale well for large-scale single cell projects (profiling of hundreds of thousands to millions of single-cells and spa^al resolu^on). WGBS is not compa^ble with simultaneous profiling of histone modifica^ons, an epigene^c modifica^on that has also been explored by the present inventors (Bartosovic et al., 2021,Bartosovic & Castelo-Branco, 2023).The prior art also offers a plurality of a^empts using fusion proteins to perform sequencing and / or30 providing libraries for profiling purposes: WO14190214 discloses a nucleic acid sequence library,wherein a protein-transposome complex binding methylated DNA is disclosed. CN117402938Adiscloses a whole genome sequencing method, using a transposon compound to fragment DNA. Forexample, it is disclosed that MBD protein is used as a binding protein to target DNA modifica^on (5mC), and that a transposase can be included in a fusion protein. WO2022 / 056309 discloses in situgenome-wide profiling. It is disclosed that an affinity reagent coupled with transposome binds a nucleosome depleted region marker, and can cleave and tag chroma^n DNA. A fusion protein of an affinity reagent and transposase is disclosed, as well as that the nucleo^de sequence of the taggedDNA segments can be determined. US201213244 discloses methods and composi^ons for selec^ve cleavage of nucleic acids with recombinant nucleases. WO2020 / 167712 discloses chroma^n mapping 40 assays and kits using long-read sequencing. WO2018 / 018008 discloses single cell whole genome libraries as well as a nucleosome-depleted nuclei. 1

[0002] Thus, prior art technologies for profiling of DNA methyla^on and DNA modifica^on are equipped withcertain limita^ons and shortages. High costs, scalability challenges and insufficient sensi^vity create aneed for further developed technical solu^ons in thefield.Summary of the inven^on The aim of the present inven^on is therefore to provide an improved method that solves at least oneof the aforemen^oned problems and drawbacks. In afirst aspect, the present inven^on refers to a method of profiling covalent DNA modifica^on at asingle-cellular or single-nuclei level comprising the steps of: (a) providing a sample solu^on, comprising cell(s) or isolated nuclei comprising a nucleus10 comprising genomic DNA, said genomic DNA comprising or is expected to comprise covalent DNA modifica^on(s); (b) adding a buffer comprising a nucleosome deple^on agent to expose the genomic DNA;(c) adding a fusion protein comprising a DNA modifica^on binding domain, having the abilityto tether the fusion protein to the DNA modifica^on and a nuclease part;(d) incuba^ng the fusion protein together with the exposed genomic DNA under condi^onsallowing the fusion protein to bind to the genomic DNA; (e) washing the solu^on of step (d) with buffer to remove any excess fusion protein, i.e.fusion protein that is unbound / untethered; (f) incuba^ng the solu^on of step (e) under condi^ons allowing the fusion protein to cut the20 genomic DNA in proximity of the DNA modifica^on(s);(g) determining the sequence of the fusion-protein incubated DNA of step (f), by high-throughput sequencing thereby iden^fying the posi^on(s) of the DNA modifica^on(s) ofthe genomic DNA that have been sequenced. In this disclosure, the inventors present an alterna^ve approach to detect genome-wide DNA methyla^on and / or other DNA modifica^on with high sensi^vity. The method is based on recogni^on of 5mC or any other DNA modifica^on by a fusion protein comprising a DNA modifica^on binding domain, and a nuclease part cu^ng the DNA. Further, the combina^on of using a nucleosomedeple^on agent and a fusion protein comprising a DNA modifica^on binding domain is a unique feature of the present disclosure. 30 Hereby, a method that is compa^ble with established droplet-based single-cell pla^orms (e.g.10X Genomics), facilita^ng the mapping of DNA methyla^on at the single-cell or single-nuclei level and holding the poten^al to achieve spa^al resolu^on, is provided. Further, the method can achieve whole-genome coverage but is cost-effec^ve, compared to the bulk methods such as Infinium Methyla^on assay and WGBS. Moreover, by using a nucleosome deple^on agent, genomic DNA is made more accessible (crucial for enhancing recogni^on by the DNA modifica^on binding agent, such as methyla^on binding domain (MBD)), while keeping nuclei intact (essen^al for the methods scalability). Thus, the purpose of nucleosome deple^on is to remove nucleosomes, but keep the nuclei intact, so they can be used for single-cell profiling or single-nuclei profiling. Further, by using a fusion protein comprising a DNA modifica^on binding domain, recogni^on of the DNA modifica^on,40 such as m5c methyla^on is facilitated. Thus, a key feature of the inven^on is the incorpora^on of nucleosome deple^on prior totransposi^on. Without this step, the MBD-Tn5 has limited access to methylated regions, and is 2

[0003] instead biased towards regions of accessible chroma^n across the genome. The present inventorsemploy nucleosome deple^on to improve the access and capture bonafide methylated loci. Therefore, this treatment is func^onally necessary to enable robust, methyla^on-targeted ac^vity of the MBD-Tn5 fusion. In some embodiments, the fusion protein has the ability to tagment the modified bases of thegenomic DNA to be profiled. In some embodiments, the nuclease part is a transposase part comprising sequencing linkers and ameans for detec^on that upon binding have the ability to be inserted to any genomic regionsenriched in modified bases of the genomic DNA to be profiled, to insert the sequencing linkers and10 the means for detec^on to the modified bases of the genomic DNA to be profiled, and to be used for determining the sequence of the fusion protein incubated DNA. In some embodiments, the nuclease part is a transposase (Tn) part which inserts sequencing linkers and a detectable barcode into the genomic DNA in proximity to the modified bases. In some embodiments, the inserted barcode uniquely iden^fies the DNA modifica^on or modality. In some embodiments the covalent DNA modifica^on to be profiled is chosen from methyla^on, preferably 5-methylcytosine (5mC).Typically, the method of the present inven^on would be used for whole genome analysis. However, profiling of parts or specific regions of the genome is also possible. In some embodiments the nucleosome deple^on agent is chosen from lithium assisted nucleosome20 deple^on (LAND) or alterna^ve detergents, such as SDS. In some preferred embodiments, thenucleosome deple^on agent is LAND. In some embodiments the DNA modifica^on binding domain of the fusion protein is a methyla^on binding domain (MBD), chosen from MBD1, MBD2, MBD3, MBD4, MBD5, MBD6, MBD7, MBD8, MBD9, MBD10, MBD11, MeCP2, BAZ2A, BAZ2B, SETB1 and SETB2. In some embodiments the DNA modifica^on binding domain of the fusion protein is MBD1 or MBD2.By using e.g. MBD1, the MBD-Tn5 fusion has been shown by the present inventors to significantlyincrease signal-to-noise ra^o and robustness of the assay compared to prior art an^body-protein Aapproaches. Also, the assay is simplified, obvia^ng the need for any intermediate an^body bindingsteps.30 In some embodiments the nuclease part of the fusion protein is a Micrococcal nuclease (MNase) orDNase I. In some embodiments the nuclease part is a transposase part (Tn), chosen from any transposase (Tn), preferably Tn5 or Tn7. In some embodiments the means for detec^on of the nuclease part comprises a unique barcode.Typically, these are 8-nucleo^de sequences, designed to iden^fy modali^es (in mul^modal profiling together with histone modifica^ons or profiling of other modifica^ons).3

[0004] In some embodiments, the barcode is a transposase-specific DNA barcode. In some embodiments themethod of the present inven^on is for prepara^on of a sequencing library comprising profiling informa^on of covalent DNA modifica^ons, such as DNA methyla^on. In some embodiments, the method is for mul^modal profiling. In some embodiments, the mul^modal profiling comprises profiling of DNA methyla^on together with open chroma^n (ATAC-seq) or histone modifica^ons. In some embodiments, the method can simultaneously detect DNA methyla^on and open chroma^n (ATAC-seq) through transposase-specific DNA barcode. In some embodiments, the method can simultaneously detect DNA methyla^on and histone 10 modifica^ons (CUT&Tag) through transposase-specific DNA barcodes. In some embodiments the method of the present inven^on is suitable for use in applica^ons relatedto single-cell resolu^on, such as on 10X Genomics, or single-cell indexing methods, such as combinatorial indexing. In some embodiments, the method of the present inven^on is suitable for use in applica^ons relatedto spa^al resolu^on, such as DBiT-sequencing or Visium, wherein mul^ple cells or regions of interestwithin a ^ssue are profiled in parallel.In a second aspect, the present inven^on relates to a kit for use in DNA methyla^on profiling atsingle-cell or single-nuclei level of genomic DNA, comprising (i) reagents for use in lithium assisted nucleosome deple^on and (ii) an MBD-nuclease fusion protein, comprising a DNA methyla^on20 binding domain and a nuclease part. In some embodiments, the MBD-Tn fusion protein is chosen from MBD1-MNase, MBD2-MNase,MBD1-DNase I, MBD2-DNase I, MBD1-Tn5, MBD1-Tn7, MBD2-Tn5 or MBD2-Tn7, for recogni^on of 5mC methylated DNA. In some embodiments, the fusion protein is an MBD1-Tn5 or MBD1-Tn7 fusion protein for recogni^on of methylated DNA. In some embodiments, the nuclease part comprises a transposase part, wherein the transposase part comprises sequencing linkers and means for detec^on, wherein the means for detec^on comprises unique barcodes. In some embodiments, the kit comprises:30 (i) a DEFND buffer for use in lithium assisted nucleosome deple^on, comprising NIBbuffer and lithium diiodosalicylate; (ii) a binding buffer, for use in incuba^on of MBD-nuclease fusion protein together witha nucleosome-depleted nuclei; (iii) an MBD-nuclease fusion protein;(iv) op^onally a wash buffer;(v) a tagmenta^on buffer;(vi) op^onally reagents for construc^on of sequencing library; and(vii) instruc^ons for use.4

[0005] In some embodiments, the kit is suitable for use in mul^modal profiling, comprising profiling of DNA methyla^on together with open chroma^n (ATAC-seq) or histone modifica^on. Thus, in some embodiments, the present inven^on is a novel method to profile DNA methyla^on using a combina^on of Lithium Assisted Nucleosome Deple^on (LAND) and methyla^on binding domain (MBD)-Tn5 transposase fusion protein. This method is poten^ally commercialized as a kit orservice. In the method, in afirst step, nucleosome deple^on, such as Lithium Assisted Nucleosome Deple^on (LAND), is used for nucleosome deple^on to make genomic DNA more accessible, whilekeeping the nuclei intact (Vitak et al., 2017). A similar approach has previously been used to perform whole genome sequencing, but not for 5mC detec^on. In the next step, the method employs a novel 10 fusion protein that were specifically designed for this purpose. The fusion protein is comprised of methyla^on binding domain (MBD) fused to Tn5 transposase (MBD-Tn5). MBD domain facilitates recogni^on of methylated DNA (5mC), whereas Tn5 transposase inserts sequencing linkers and a barcode into genomic DNA through cut and paste mechanisms. Nucleosome deple^on treatment, such as LAND treatment, is crucial for enhancing the recogni^on of 5mC by MBD-Tn5 fusion proteins within the genomic DNA. Maintaining intact nuclei is also essen^al for the method's scalability. The method is compa^ble with established droplet-based single-cell pla^orms (e.g.10x Genomics), facilita^ng the mapping of DNA methyla^on at the single-cell level and holding the poten^al to achieve spa^al resolu^on. This method of the inven^on can yield data of comparable quality to that of WGBS but the 20 sequencing depth requirement and cost is much less. With the growing demands on single-cell and spa^al analyses in the biomedicalfield, the scalability of this technique aligns with the requirements of a lot of researchers in relatedfields. The principles of the method of the inven^on can be used to develop kit(s) for DNA methyla^onsequencing library preps in bulk, single-cell, and / or spa^al analysis. The method can also be used as aservice to support the kits. Thus, the present inven^on is unique in that it introduces the combina^on of (1) a methyla^on-binding fusion transposase, and (2) a chroma^n prepara^on strategy to remove nucleosomes. Thiscombina^on provides a synergis^c effect which is both non-obvious and func^onally advantageous. Effects and features of the second aspect are to a large extent analogous to those described above in 30 connec^on with thefirst aspect. Embodiments men^oned in rela^on to thefirst aspect are largely compa^ble with the second aspect. The present disclosure will become apparent from the detailed descrip^on given below. The detailed descrip^on and specific examples disclose preferred embodiments of the disclosure by way of illustra^on only. Those skilled in the art understand from the guidance in the detailed descrip^on thatchanges and modifica^ons may be made within the scope of the disclosure. Hence, it is to be understood that the herein disclosed disclosure is not limited to the par^cular component parts of the product described or steps of the methods described since such product andmethod may vary. It is also to be understood that the terminology used herein is for the purpose of describing par^cular embodiments only, and is not intended to be limi^ng. It should be noted that, as 40 used in the specifica^on and the appended claim, the ar^cles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements unless the context explicitly dictates 5

[0006] otherwise. Thus, for example, reference to "a unit" or "the unit" may include several devices, and the like. Furthermore, the words "comprising", "including", "containing" and similar wordings do not exclude other elements or steps. Brief descrip^on of the drawings Figure 1: Genome browser view shows DNA methyla^on profiles obtained by MBD-Tn5 withoutLAND, MBD-Tn5 with LAND, and WGBS. Figure 2: Sequencing depth required for MBD-Tn5 with LAND and WGBS methods to obtain the data shown in Figure 1. Figure 3: Schema^c view of the method of the present inven^on. (a) LAND and (b) MBD-Tn510 incuba^on. Figure 4: Example of sequencing library structure. Figure 5: Library structure ((a)-(h)).Defini^ons The term “single-cell level” refers to the level of an individual cell, i.e. analysis of the single-cell level refers to evalua^on of the individual cell. “Single-cell resolu^on” thus provides informa^on about the individual cells, and “single-cell sequencing” examines the nucleic acid sequence informa^on from individual cells In analogy, the term “single-nuclei level” refers to the level of an individual cell nucleus.The term “spa^al” refers to a specific area or a region within a ^ssue, and thus to informa^on (such 20 as gene expression informa^on) obtained from such areas or regions, e.g. informa^on about cell-cell interac^ons. The term “CpG” refers to “5’-C-phosphate-G-3’”, that is, cytosine and guanine separated by only one phosphate group. “CpG islands” (“CGIs”) refer to regions of the genome that contain a large number of CpG dinucleo^de repeats. CGIs are usually associated with lack of DNA methyla^on, and thus “CpG density” is inversely correlated with DNA methyla^on. The term “profiling”, in the context of this disclosure, refers to the process of obtaining a specific DNA pa^ern (a “profile”) from a sample of cells or ^ssue of a person, pa^ent and / or ^ssue. The term “tagmenta^on” refers to an ini^al step in DNA library prepara^on where unfragmentedDNA is cleaved and tagged for analysis. Typically, such cleavage and tagging can be performed by30 using an enzyme or fusion protein having transposase ac^vity. In the context of the present disclosure, a “nuclease” includes any enzyme having the capacity to cleave / cut a DNA molecule, whereas “transposase” refers to a nuclease also having the capacity to insert a sequence and / or to be inserted into the DNA molecule cleaved. Thus, a transposase has both a nuclease ac^vity and an inser^on / tagging ac^vity. The term “high-throughput sequencing” refers to technologies that sequence DNA (and RNA) in a rapid and cost-effec^ve manner, also known as next-genera^on sequencing. Any sequencing methods exemplified in this disclosure is included in this concept. 6

[0007] The terms “solu^on” or “suspension” with regard to the solu^ons / suspensions containing cell material and buffers etc according to the present disclosure may be used interchangeably in this disclosure. “NIB buffer” refers to a nuclei isola^on buffer.“DEFND buffer” refers to a specific LAND buffer. Detailed descrip^on of the inven^on The present inven^on thus provides methods and kits for improved DNA modifica^on profiling at a single-cellular, single-nuclei or spa^al level, wherein the combina^on of (i) using a nucleosome deple^on reagent, thereby making the genomic DNA more accessible, while keeping the nuclei intact,10 with (ii) incuba^ng the genomic DNA with a fusion protein comprising a modifica^on binding domainand a nuclease part, allowing binding and subsequent inser^on of suitable sequencing linkers and means for detec^on, results in advantages such as higher sensi^vity and improved scalabilitycompared to prior art methods. The method In afirst aspect, also referring tofigure 3, a sample solu^on comprising cell(s) and / or nuclei to beanalysed is provided, which cells comprises a nucleus comprising genomic DNA. The cells are typicallyof eukaryo^c origin, such as plant or animal cells, such as human cells or mouse cells. Any types of^ssues may be used, such as brain heart, muscle, lung and blood. Typically, the cells are provided in asolu^on and / or in any form that can be solubilized. 20 The genomic DNA of the cells contain or are expected to contain covalent DNA modifica^ons in at least a part of the genomic DNA. Typically, the profiling process is made for the whole genome, and in some embodiments, it is concentrated to a part or region of the genomic DNA that are of specialinterest. The DNA modifica^ons can be chosen from various type of modifica^ons, such as methyla^on, including 5-methylcytosine modifica^on. However, other modified bases exist in DNA, for example 5-hmC, 5-fC,6-mA. Any DNA modifica^on might be detected by the principle of the present inven^on, e.g. LAND + fusion of 5-hmC / 5-fC / 6-mA reader to Tn5. Examples of hmC readers are for exampleUhrf2, Thy28 and Wdr76 (see also h . Examplesof 6mA readers are YTHDF1, YTHDF2 and YTHDC5.30 To the sample solu^on is added, in a next step, a buffer comprising a nucleosome deple^on agentthereby forming a cell suspension. The nucleosome deple^on agent has the capacity to remove the nucleosomes, make the genomic DNA more accessible, while keeping the nuclei intact. The nucleosome deple^on agent can be chosen from any agent having the desired effect, such as sodium dodecyl sulfate (SDS). However, a preferred choice having shown a strong effect is lithiumassisted nucleosome deple^on agent (LAND). Thus, typically the nucleosome deple^on agent ischosen from lithium assisted nucleosome deple^on (LAND) or alterna^ve detergents, such as SDS, preferably LAND. For the LAND, it is also possible to modify the composi^on of the buffer, usedifferent concentra^ons of lithium diiodosalicylate, and incuba^on ^mes to remove nucleosomes and maintain nuclei intact. In some embodiments, a concentra^on of lithium diiodosalicylate in the40 interval of 1 mM to 50 mM can be used, such as about 12,5 mM. In some embodiments, the 7

[0008] incuba^on ^me, e.g. when using a working concentra^on of 12,5 mM, can be in the interval of 30 seconds to 10 minutes, such as about 5 minutes. Further, there are other ways to deplete nucleosomes from nuclei using alterna^ve detergents, for example cross-linking and SDS treatment, but other alterna^ves might exist as well. However, before SDS treatment, the nuclei need to befixedusing formaldehyde. As the next step, a specifically designed fusion protein is added to and incubated with the suspension, so that the modifica^on binding domain of the fusion protein is allowed to bind to the exposed genomic DNA, typically the nuclei. This incuba^on is typically performed overnight, and canalso be done for about 10 minutes or for a few hours, The temperature is typically about 4^C, and can10 also be up to about room temperature, as long as proper binding is achieved. The incuba^on can be performed as exemplified in the Example sec^on. Typically, incuba^on is performed in a horizontal roller at 4 degrees overnight. However, other temperatures and incuba^on condi^ons could also work, as long as the fusion protein is allowed to bind to the DNA modifica^onsites that are available and to be profiled. For example, the incuba^on can be performed at room temperature at a ^me interval ranging from 10 minutes to 2 hours. Other non-extreme temperatures lower than 4 C and higher than room temperature will also typically work and are included (as long asthe solu^on (water) is not below the freezing point, and remains in a liquid phase). The fusion protein comprises at least two parts: one part comprising a DNA modifica^on binding func^onality, so that the fusion protein binds to the DNA modifica^ons to be profiled. These DNA 20 modifica^ons could be of any type, such as 5-methylcytosine methyla^on (5mC), for which type of modifica^on the present inven^on is especially suited. In such case, the DNA modifica^on binding part is a methyla^on binding domain (MBD), such as a methyla^on binding domain chosen fromMBD1, MBD2, MBD3, MBD4, MBD5, MBD6, MBD7, MBD8, MBD9, MBD10, MBD11, MeCP2, BAZ2A, BAZ2B, SETB1 and SETB2, preferably MBD1 or MBD2. There are currently 11 MBD domain proteinsthat have a bit different sequence of MBD domains. It is possible to use any MBD domains to engineer MBD-Tn fusion proteins, such as MBD1-Tn5 and generate good quality data. In addi^on to the DNA modifica^on binding part, the fusion protein also comprises a nuclease part, that comprises a nuclease, that has the ability to cut the genomic DNA. In some embodiments, forexample when the nuclease is a transposase, the nuclease part (or transposase part) typically30 comprises sequencing linkers and means for detec^on, whereby the transposase part has the abilityto break the genomic DNA and insert the sequencing linkers and means for detec^on into thegenomic DNA, at or close to the posi^on of a DNA modifica^on, thereby facilita^ng recogni^on of the DNA modified posi^on. The nuclease part can be any nuclease having the desired effect. However, a transposase (Tn) has been shown to be especially advantageous for the purposes of the present inven^on, especially when profiling methyla^ons. Typically, the nuclease is chosen from anytransposase (Tn) or nuclease having similar func^on, such as Micrococcal nuclease (MNase) or DNaseI, or a transposase chosen from Tn5 or Tn7. In some embodiments, for MBD-Tn fusion proteins, it isalso an op^on to fuse the MBD domain and other Transposases such as Tn5 or Tn7 or other nucleasessuch as MNase or DNase I to have similar results. Fusing MBD-Tn5 with more domains such as a DNA- 40 binding domain of transcrip^on factors (DBD) is also expected to be able to detect transcrip^on factor binding sites, without the effects of chroma^n context. While not a focus of the presentinven^on, fusing MBD-Tn5 with other DNA-binding domains (e.g., transcrip^on factor binding domains) is also contemplated for detec^ng other genomic features (like transcrip^on factor binding 8

[0009] sites). When a nuclease not having a transposase func^on is used, i.e. a nuclease such as MNase orDNase I, any sequences or reagents necessary for sequencing, as well as any barcodes, must be added in the downstream process, as a non-transposase nuclease typically do not have the ability to introduce any sequence upon cleaving the DNA molecule. Thus, the fusion protein can comprise a nuclease having the capacity to cleave (cut) the DNA molecule at certain posi^ons, or it can comprise a nuclease having transposase ac^vity, i.e. a transposase that has the capacity to cut and paste the DNA molecule at certain posi^ons, i.e. it basically does two things at the same ^me. When using a nuclease without transposase ac^vity, the cut pieces of DNA can be purified, but sequencing linkers have to be purified and linkers ligated by10 e.g. DNA ligase. Hence, a more complex workflow must be followed by using a nuclease.A^er having incubated the fusion protein together with the exposed genomic DNA, in order to have the modifica^on binding domain of the fusion protein to bind to, or close to, modifica^ons occurring in the genomic DNA (i.e. modifica^on that are specific for the modifica^on binding domain (such as m5c methyla^ons, histone modifica^ons etc.)), the solu^on of fusion protein together with exposed genomic DNA is washed to remove any excess fusion protein. Typically, the washing procedure isperformed at least twice. Thus, before the tagmenta^on, the suspension including fusion protein is washed to remove excess fusion protein that is unbound / untethered to the DNA. A tagmenta^on step is then performed, wherein the fusion protein is incubated with the cell 20 suspension that has undergone nucleosome deple^on, so that the fusion protein can cut the DNA and insert the sequencing linkers and means for detec^on at the posi^ons of the modifica^on to be profiled. During tagmenta^on, the washed solu^on is incubated under condi^ons ac^va^ng the nuclease part of the fusion protein, thereby allowing the fusion protein to cut the genomic DNA in proximity of the DNA modifica^on(s). This incuba^on step is also referred to as tagmenta^on in some embodiments of the present disclosure. Typically, the tagmenta^on step is performed at a higher temperature thanin the previous incuba^on, typically at about 37^C. However, varia^ons may occur, as long as condi^ons ac^va^ng the nuclease / transposase func^on of cu^ng or cu^ng and pas^ng the DNA is obtained. The fusion protein cuts the genomic DNA in proximity of the DNA modifica^on(s) to which 30 it has bound, which typically is within about 50-100 bp from the modifica^on(s), so that for example about 150 bp fragments with high modifica^on enrichment are obtained. Thus, typically the posi^on is approximate with + / - a few hundred base pairs, thereby showing enrichment and typically not theexact posi^ons of the modifica^on(s). The same principles apply to mul^modal profiling, i.e. regionsof enrichment of each modifica^on are shown. The sequencing linkers are used for PCR amplifica^on of the library. Examples of the PCRamplifica^on procedure and sequencing linkers adapted for use with Tn5 is provided in the Examplesec^on. Typically, one part of the sequencing linkers is a so-called mosaic-end adapter, which isrequired for recogni^on by the transposase, such as Tn5. The second part of the sequencing linker serves for sequencing primers annealing. Typically, the means for detec^on, which in this context 40 comprises iden^fica^on of modality (DNA methyla^on, open chroma^n or histone modifica^on), such as barcodes, can be posi^oned in between these sequencing linker parts. 9

[0010] The means for detec^on can be any molecule or part that provide the desired effect of allowing detec^on and / or iden^fica^on of the profiled DNA modifica^on(s). Typically, the means for detec^on is integrated in the nuclease part in the form of a unique barcode. The unique barcode may for example be posi^oned in between the parts of the sequencing linker. Typically, the unique barcodes are cons^tuted of 8-nucleo^de sequences, designed to iden^fy modali^es (in mul^modal profiling together with histone modifica^ons). However, varia^ons are included. For example, in a technicalperspec^ve, any length that allows to dis^nguish modali^es, typically 6-8 nucleo^des, and in some embodiments as short as 2 nucleo^des or as long as 15 or 20 nucleo^des, or even longer, are also included. 10 As afinal step, the sequence of the genomic DNA at posi^ons where the fusion protein has inserted sequencing linkers is determined, thereby iden^fying the posi^ons of the genomic DNA includingmodifica^ons of the type that is intended to be iden^fied. This can be achieved as exemplified in theExample sec^on. Other available sequencing methods may also be used, including Illumina short readsequencing, which is primarily used by the present inventors, and MGI short read, nanopore long read, pac bio long read sequencing, and similar sequencing methods, including high-throughput sequencing methods and / or next genera^on sequencing (NGS) methods. Applica^ons The method and kit of the inven^on can be used in various applica^ons related to profiling of DNA modifica^ons, such as methyla^on, at a single-cellular, single-nuclei and / or spa^al level. 20 In some embodiments, the method of the inven^on is used for prepara^on of a sequencing library comprising profiling informa^on of covalent DNA modifica^ons, such as DNA methyla^ons. In some embodiments, the method and kit of the inven^on is used for applica^ons related to single- cell resolu^on, such as on 10X Genomics, or single-cell indexing methods, such as combinatorial indexing. However, other applica^on related to single-cell resolu^on are also included. In some embodiments, the method and kit of the inven^on is used for applica^ons related to spa^alresolu^on. Spa^al resolu^on may for example be accomplished using DBiT-seq(h^ps: / / www.nature.com / ar^cles / s41586-023-05795-1) or other spa^al methods, such as Visium, wherein mul^ple cells are profiled in parallel. However, other applica^ons related to spa^alresolu^on are also included. 30 In some embodiments, the method is used for mul^modal profiling. In some embodiments, the mul^modal profiling comprises profiling of DNA methyla^on together with open chroma^n (ATAC- seq) or histone modifica^ons. More than two modes are possible to profile using the method of the inven^on. Other modifica^on alterna^ves than those listed here are also included. For example, atleast two 2 histone modifica^ons could be profiled simultaneously, and in some embodiments up to10, 20, 30, 40 or 50 simultaneous modifica^ons. The upper theore^cal limit would be defined by crowding of the Tn5 / Tn7 inser^ons in the DNA in case of overlapping features / modali^es.Applica^ons related to single-nuclei resolu^on are also included using the method and kit of thepresent inven^on. The kit 10

[0011] In a second aspect, the present inven^on refers to a kit for use in DNA methyla^on profiling at single-cell or single-nuclei level of genomic DNA. The kit would typically comprise the components / ingredients that are necessary for performing the method for any sample, at least including (i) reagents for use in lithium assisted nucleosome deple^on and (ii) a MBD-nuclease fusion protein, comprising a DNA methyla^on binding domain and a nuclease part. In some embodiments, the fusion protein is chosen from fusion proteins that are based on MBD1 or MBD2 together with a Tn part chosen from Tn5 or Tn7, or a nuclease part chosen from MNase or DNase I, thereby allowing recogni^on of m5c methylated DNA. In some embodiments of the kit, the nuclease part comprises a transposase part, wherein the 10 transposase part comprises sequencing linkers and means for detec^on, wherein the means for detec^on comprises unique barcodes, as disclosed and discussed in other parts of this applica^on. Such sequencing linkers and barcodes are included here. In some embodiments, the kit would include addi^onal components and / or ingredients, such as:- a DEFND buffer for use in lithium assisted nucleosome deple^on, comprising NIB buffer andlithium diiodosalicylate; -a binding buffer (also called an^body buffer in the context of this disclosure), for use inincuba^on of MBD-nuclease fusion protein together with a nucleosome-depleted nuclei;- an MBD-nuclease fusion protein;- a wash buffer, for washing away non-binding / excess fusion protein;20 - a tagmenta^on buffer;- op^onally reagents for sequencing; and- instruc^ons for use.Various modifica^ons of volume, concentra^on and other parameters are within the scope of the present disclosure, as long as the overall objects of the inven^on are achieved. Further, in some embodiments, the kit would be adapted for mul^modal profiling. comprising profiling of DNA methyla^on together with open chroma^n (ATAC-seq) or histone modifica^ons. Some components / ingredients would be generic, i.e. to be used for any sample, regardless of cell type, modifica^on type and genomic DNA sequences. This would typically include buffers to be used as well as MBD-nuclease fusion protein including sequencing linkers and unique barcodes (means for30 detec^on). Also, reagents for sequencing would be generic, as long as informa^on on the specific sample DNA sequences is not required. Some components / ingredients could however be specific, i.e. dependent on the specific sample and / or its genomic DNA sequence to be profiled, and may therefore not be suitable to be included in a generic kit, but could be included in a kit adapted forprofiling of a specific type of sample, cell, modifica^on type and / or genomic DNA. The present disclosure will now be described with reference to the following examples. EXAMPLES Example 1 - Lithium-assisted nucleosome deple^on (LAND) followed by Tn5-based DNAmethyla^on profiling 11

[0012] Buffer prepara^on NIB buffer (for LAND), prepare fresh 100 mM lithium diiodosalicylate (396 g / mol): Weigh 7.92 mg of lithium diiodosalicylate and dissolvein 200 μl Nuclease-free Water. Weigh chemicals first and add water later in a proportion. Stocksolution can be stored at -20 C.DEFND buffer DEFND buffer comprises 175 μl NIB and 25 μl 100 mM lithium diiodosalicylate.2X Wash buffer 10 Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 13 μL 2 M spermidine, add 1 Roche Complete Protease Inhibitor EDTA-Free tablet, bring the final volume to 25 mL with nuclease free water. Store the bufferat 4 °C for up to 1 week. Antibody (binding) buffer Mix 8 μL 0.5 M EDTA, 20 μL Digitonin 5%, 2 μL of 10% NP-40 and 200 μL 20% BSA with 1 mL 2X Washbuffer and chill on ice. Make fresh. Volume needed is 300 μL per sample (200 μL for nuclei extraction +100 μL for antibody incuba^on).Prepare 500 μL per sample to have extra. 12

[0013] Digitonin300-Wash Mix 1 mL 2X Wash Buffer, 60 μL 5 M NaCl, 20 μL Digitonin 5%, 2 μL of 10% NP-40, 200 μL 20% BSA, bring thefinal volume to 2 mL with nuclease free water. Make fresh. Prepare 500 μL per sample. * 150mM in wash + 150mM supplemented here Tagmentation buffer Mix 750 μL 2X Wash buffer, 15 μL 1 M MgCl2 (to 10 mM), 45 μL 5 M NaCl, 15 μL 5% Digitonin, 1.5 μL10 of 10% NP-40, 150 μL 20% BSA, bring the final volume to 1.5 mL with nuclease free water. Makefresh. Prepare 250 μL per sample. 13

[0014] 2X TD buffer Mix 20 μL 1M Tris pH 7.5, 10 μL 1 M MgCl2 (to 10 mM), 200 μL 100% dimethylformamide, bring the final volume to 1 mL with dH2O. Store the buffer at -20 °C for up to 1 year. Detailed protocol 1. LAND treatmentTip: Each LAND-treated sample requires 200,000 cells, If processing more samples, pool samples together, scale up buffers (e.g.3 samples using 600 μl DEFND buffer but NIB buffer can be 10 ml),10 and split cells at step f (e.g.3 samples using approximately 330 μl antibody buffer forresuspension). a) Pellet 200,000 cells and wash with ice-cold PBS (300 g for 5 minutes at 4 degrees) in 1.5 mlEppendorf tubes b) Resuspend in 200 µL of DEFND buffer (175 µL NIB, 25 µL 100 mM lithium diiodosalicylate)and incubate on ice for 5 minutes c) Immediately add 10 ml NIB buffer and then centrifuge at 4 °C for 5 minutes at 300 gd) Resuspend in 200 μl antibody buffere) Transfer into 0.5 ml tubes and centrifuge for 5 mins at 300 gf) Resuspend in 100 μl antibody buffer20 2. Fusion protein incubationa) Add 1 µL MBD fusion proteins loaded with specific barcodes (such as MBD1-Tn5 Barcode A)into the 100 μl nuclei suspension and mix thoroughly.b) Place the tubes on a horizontal roller and incubate at 4°C overnight.3. Nuclei washing and Tn5 (P5) tagmentationa) Next day, centrifuge for 3 min at 600g at 4°C and discard the supernatant. Use a swinging30 bucket rotor centrifuge for all nuclei handling steps. b) Resuspend the nuclei in 200 µL of Dig-300 wash buffer.c) Repeat previous steps for a total of 2 washes and remove the supernatant carefully.d) Resuspend the nuclei pellet in 200 µL of Tagmentation buffer. Pipette mix five times with 200μL pipette tip to resuspend gently the pellet. 14

[0015] e) Incubate at 37°C for 1 h at a heater block. At 30 minutes of the 1 h incubation pipette mix toprevent nuclei sedimentation. If there are no suitable adapters for 0.5 ml tubes in the thermomixer, use 1.5ml microcentrifuge tubes with ~800μl of water as adapters or use awater bath instead. f) Remove the samples from the thermomixer and centrifuge for 3 min at 600g at 4°C.g) Remove 100 µL of supernatant4. Tagmented gDNA purification using Zymo kit10 a) Add 500 μL DNA binding buffer to the tube (Zymo DNA Clean & Concentrator-5) and transferthe mix into zymo kit column (pre-assembled with bottom waste collection tube) b) Centrifuge for 1min at 12,000 xg. Discard the flow-through.c) Add 200 µL of zymo wash buffer to the column. Centrifuge for 1 min at 12,000 xg. Discard thesupernatant. d) Repeat the wash step once more.e) Perform dry spin – After discarding the supernatant, centrifuge the assembled columns oncemore for 1 minute at 12,000 xg f) Transfer the column into a clean 1.5ml eppendorf tube. Discard the waste collection tube.g) Add 25 µL of zymo elution buffer (EB) and incubate for 2 minutes at RT.20 h) Centrifuge for 1min at 12,000 xg. Discard the column and save the flow-through.i) Measure DNA concentration with Qubit High Sensitivity using 1 μL of the purified DNA.j) Expected yield of gDNA at this step is between 4-7 ng / μl regardless of epitope.5. Linear Amplificationa) Prepare the following master mix for linear amplification in a PCR tube. For multiple samples,prepare a master mix. 15

[0016] Note: Use 10 µL of gDNA for histone marks. In case of less-abundant epitopes, use all of the gDNA from Step 4. b) Run the following program in a PCR cycler (Same as 10x Genomics scATAC-seq kit BarcodingPCR program) 6. Perform PCR clean up with 1.2x SPRI bead purification.10 a) Add 60 μL of SPRI beads to the PCR reaction and mix by pipetting up-down 10x.b) Incubate for 10 minutes at RT.c) Place the tubes with beads on magnet, wait ~2 minutes until the beads are completelyattached to the side of the tube. d) With tubes still on magnet, remove the supernatant.e) Add to each tube 200 μL of 80% Ethanol. Incubate for 30s.f) With tubes still on magnet, remove the ethanol wash.g) Repeat the wash once more for total of 2 washes.h) Quickly spin the beads in benchtop microfuge and place back on the magnet.i) Remove the residual ethanol with P10 pipette.20 j) Remove the tubes from the magnet and resuspend them in 20 μL of nuclease-free water.Incubate on table for 2 minutes. k) Place the tubes on magnet and wait for the beads to attach to it.l) Collect the supernatant to a clean tube.m) Measure the Linear amplification product yield with a Qubit dsDNA HS kit following themanufacturer´s instructions. Note: Expected yield of LA product is between 40-100 ng (2-5 ng / μl) if using 10 μl of H3K27me3-tagemented template and 200,000 cells as starting material. 16

[0017] 7. Use (up to) 50 ng of LA product for Tn5-ME-B transposition (P7).50 ng of the LA product is best to be used in libraries to be sequenced. If the experiment is foroptimisations or ME-B Tn5 titration, 20ng LA product is also fine. Transposition reaction: Mix the following reaction: 10 * Exact ratio of P7 Tn5 to LA product needs to be determined for each P7 Tn5 loading a) Incubate in a PCR cycler for 30 min at 37°C and hold at 4°C. Set the heated lid at 50°C.b) Add 200 μL DNA binding buffer (Zymo DNA Clean & Concentrator-5) and perform DNApurification with 2x 200 μL washes and one dry spin (as outlined in steps 20-27). Perform the elution in 20 μL of EB. c) Prepare the following master mix for library amplification 17

[0018] 10X_LA_primer_noBCD (10 μM) a) Run the following program in a PCR cycler *Number of PCR cycles can be determined by using 1 uL of P7-tagmented product in qPCR8. Library purification using SPRI beadsa) Purify the product using 1.2x SPRI beads as described before.10 b) Measure the library yield with a dsDNA-specific assay, such as Qubit DNA HS following themanufacturer's instructions. c) Verify the size distribution of the library by a capillary electrophoresis (e.g AgilentBioanalyzer High Sensitivity kit following manufacturer's instructions). Identify the optimalTn5-MeB ratio to DNA that gives the best library distribution (with a range of 300-700 bp). Example 2 – Co-profiling of two histone marks and DNA methyla^onNuclei isola^on and an^body incuba^on (Buffers used are as provided in Example 1)20 a) Transfer 200,000 cells per reac^on into a 1ml microcentrifuge tubeb) Centrifuge for 5 min at 300g at 4°C. Remove supernatant and resuspend the cell pellet in cold(4°C) 1x PBS. 18

[0019] c) Distribute cells into individual 0.5ml tubes per experimentd) Centrifuge the tubes for 5 min at 300g at 4°C.e) Resuspend gently the cell pellets in each tube in 200 µL of ice-cold an^body buffer.f) Incubate the mixture on ice for 3 min for nuclei extrac^on.g) Centrifuge for 3 min at 600g at 4°C and discard the supernatant.h) During the centrifuga^on, mix 100 µL of an^body buffer with 1 µL (1:100) of two primaryan^bodies raised in mouse and rabbit (for example rabbit H3K27ac (Ab177178) and mouse H3K27me3 (Ab6002)) and 1 µL (1:100) of an^-rabbit nano-Tn5 and 1µl of (1:100) an^-mouse nano-Tn5 loaded with different barcodes into each tube. Thus, the different barcodes are10 used to differen^ate histone marks (e.g., H3K27ac and H3K27Me3). i) Place the tubes on a horizontal roller and incubate at 4°C overnight.Nuclei washing and Tn5 (P5) tagmenta^on a) Next day, centrifuge for 3 min at 600g at 4°C and discard the supernatant. Use a swingingbucket rotor centrifuge for all nuclei handling steps. b) Resuspend the nuclei in 200 µL of Dig-300 wash buffer.c) Repeat previous steps for a total of 2 washes and remove the supernatant carefully.d) Resuspend the nuclei pellet in 200 µL of Tagmenta^on buffer. Pipe^e mixfive ^mes with 20020 μL pipe^e ^p to resuspend gently the pellet. e) Incubate at 37°C for 1 h at a heater block. At 30 minutes of the 1 h incuba^on pipe^e mix toprevent nuclei sedimenta^on. If there are no suitable adapters for 0.5 ml tubes in the thermomixer, use 1.5ml microcentrifuge tubes with ~800μl of water as adapters or use awater bath instead. LAND treatment a) A^er 1h tagmenta^on, remove the samples from the thermomixer and centrifuge for 3 minat 600g at 4°C.30 b) Resuspend in 200 µL of DEFND buffer (175 µL NIB, 25 µL 100 mM lithium diiodosalicylate)and incubate on ice for 5 minutes. c) Immediately add 10 ml NIB buffer and the centrifuge at 4 °C for 5 minutes at 300 gd) Resuspend in 200 μl an^body buffere) Transfer into 0.5 ml tubes and centrifuge for 5 mins at 300 g at 4 °C.f) Resuspend in 100 μl an^body bufferThe following steps are the same as in the previous example 1, i.e. from the fusion protein incuba^on to thefinal library purifica^on using SPRI beads. Thus, con^nue with Step 2 – 8 in Example 1 -Lithium-assisted nucleosome deple^on (LAND) followed by Tn5-based DNA methyla^on profiling. 40 Example 3 The inventors have compared the DNA methylation profiles obtained by the combination of MBD-Tn5 without LAND (track 1) and with LAND (track 2) to those of WGBS (track 3) in K562 cells (Figure 1). MBD-Tn5 + LAND method recapitulates the DNA methylation pattern obtained by WGBS in human 19

[0020] cells. MBD-Tn5 and LAND method only need 5 – 30 million reads to obtain a similar DNA methylationpattern as WGBS which requires at least 500 million reads (Figure 2). Example 4 – Fusion protein sequencesBelow are examples of fusion proteins used in the context of the present invention disclosed. Sequencing linkers and barcodes are not included here (but can be seen as part of the DNA sequences in Example 5): 10 20 30 Linker (SEQ ID NO: 5): GGGGSGGGGSGGGGSGGGGSFor the following fusion protein sequences, the corresponding parts (His + 3xFLAG tag (SEQ ID NO: 2),Tn5 transposase (SEQ ID NO:4) and linker (SEQ ID NO: 5)) apply as for psfMeCP2Tn5-c001. Themodifica^on binding domain varies between the fusion proteins.psfMBD2Tn5-c001 (SEQ ID NO: 6) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKATESGKRMDCPALPPGWKKEEVIRKSGLSA GKSDVYYFSPSGKKFRSKPQLARYLGNTVDLSSFDFRTGKMMPSKLQKGGGGSGGGGSGGGGSGGGGSHMITSAL 40 HRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQ TVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPAD ADEKESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQ PELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPV ESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGL LKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEAL QSKLDGFLAAKDLMAQGIKI MBD domain (MBD2) (SEQ ID NO: 7):ATESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVDLSSFDFRTGKMMPSK LQK 50 psfMBD1_Tn5-c001 (SEQ ID NO: 8) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTAEDWLDCPALGPGWKRREVFRKSGATCG RSDTYYQSPTGDRIRSKVELTRYLGPACDLTLFDFKQGILGSGGSGGTGSGGTGSGGSGTSMITSALHRAADWAK 20

[0021] SVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEF PELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGK WLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQI SIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALR VIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKEAEHVE SQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFL AAKDLMAQGIKI MBD domain (MBD1) (SEQ ID NO: 9):GTAEDWLDCPALGPGWKRREVFRKSGATCGRSDTYYQSPTGDRIRSKVELTRYLGPACDLTLFDFKQGILGS 10 psfMBD4_Tn5-c001 (SEQ ID NO: 10) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTRKSVPCGWERVVKQRLFGKTAGRFDVYF ISPQGLKFRSKSSLANYLHKNGETSLKPEDFDFTVLSKGSGGSGGTGSGGTGSGGSGTSMITSALHRAADWAKSV FSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPE LLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWL AAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQISI PQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVI DIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKEAEHVESQ SAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFLAA KDLMAQGIKI 20 MBD domain (MBD4) (SEQ ID NO: 11):GTRKSVPCGWERVVKQRLFGKTAGRFDVYFISPQGLKFRSKSSLANYLHKNGETSLKPEDFDFTVLSK psfMBD5_Tn5-c001 (SEQ ID NO: 12) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTDKEGGLPAIQVPVGWQRRVDQNGVLYVS PSGSLLSCLEQVKTYLLTDGTCKCGLECPLILPKVFNFDPGAAGSGGSGGTGSGGTGSGGSGTSMITSALHRAAD WAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLA QEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKE SGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGG 30 YQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQ ALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKEAE HVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLD GFLAAKDLMAQGIKI MBD domain (MBD5) (SEQ ID NO: 13):GTDKEGGLPAIQVPVGWQRRVDQNGVLYVSPSGSLLSCLEQVKTYLLTDGTCKCGLECPLILPKVFNFDPGAA psfBAZ2A_Tn5-c001 (SEQ ID NO: 14) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTIATPEEVRLPLQHGWRREVRIKKGSHRW QGETWYYGPCGKRMKQFPEVIKYLSRNVVHSVRREHFSFSPRMPGSGGSGGTGSGGTGSGGSGTSMITSALHRAA DWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKL 40 AQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEK ESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELG GYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLA QALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKEA EHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKL DGFLAAKDLMAQGIKI MBD domain (BAZ2A) (SEQ ID NO: 15):GTIATPEEVRLPLQHGWRREVRIKKGSHRWQGETWYYGPCGKRMKQFPEVIKYLSRNVVHSVRREHFSFSPRMP psfBAZ2B_Tn5-c001 (SEQ ID NO: 16) 50 MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTVTDERELRIPLEYGWQRETRIRNFGGRL QGEVAYYAPCGKKLRQYPEVIKYLSRNGIMDISRDNFSFSAKIRGSGGSGGTGSGGTGSGGSGTSMITSALHRAA DWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKL AQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEK ESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELG GYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLA 21

[0022] QALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKEA EHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKL DGFLAAKDLMAQGIKI MBD domain (BAZ2B) (SEQ ID NO: 17):GTVTDERELRIPLEYGWQRETRIRNFGGRLQGEVAYYAPCGKKLRQYPEVIKYLSRNGIMDISRDNFSFSAKIR psfMBD3_Tn5-c001 (SEQ ID NO: 18) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTMERKRWECPALPQGWEREEVPRRSGLSA GHRDVFYYSPSGKKFRSKPQLARYLGGSMDLSTFDFRTGKMLMSGSGGSGGTGSGGTGSGGSGTSMITSALHRAA 10 DWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKL AQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEK ESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELG GYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLA QALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKEA EHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKL DGFLAAKDLMAQGIKI MBD domain (MBD3) (SEQ ID NO: 19):GTMERKRWECPALPQGWEREEVPRRSGLSAGHRDVFYYSPSGKKFRSKPQLARYLGGSMDLSTFDFRTGKMLMS 20 psfMBD6_Tn5-c001 (SEQ ID NO: 20) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTDRAGGPVATSVPIGWQRCVREGAVLYIS PSGTELSSLEQTRSYLLSDGTCKCGLECPLNVPKVFNFDPLAPGSGGSGGTGSGGTGSGGSGTSMITSALHRAAD WAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLA QEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKE SGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGG YQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQ ALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKEAE HVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLD GFLAAKDLMAQGIKI30 MBD domain (MBD6) (SEQ ID NO: 21):GTDRAGGPVATSVPIGWQRCVREGAVLYISPSGTELSSLEQTRSYLLSDGTCKCGLECPLNVPKVFNFDPLAP psfSETB1_Tn5-c001 (SEQ ID NO: 22) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTYRGKNPLLVPLLYDFRRMTARRRVNRKM GFHVIYKTPCGLCLRTMQEIERYLFETGCDFLFLEMFCLDPYVLGSGGSGGTGSGGTGSGGSGTSMITSALHRAA DWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKL AQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEK ESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELG GYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLA 40 QALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKEA EHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKL DGFLAAKDLMAQGIKI MBD domain (SETB1) (SEQ ID NO: 23):GTYRGKNPLLVPLLYDFRRMTARRRVNRKMGFHVIYKTPCGLCLRTMQEIERYLFETGCDFLFLEMFCLDPYVL psfSETB2_Tn5-c001 (SEQ ID NO: 24) MHHHHHHSSGVDLGTENLYFQSGDYKDHDGDYKDHDIDYKDDDDKGTLNLKGENPLQLPIKCHFQRRHAKTNSHS SALHVSYKTPCGRSLRNVEEVFRYLLETECNFLFTDNFSFNTYVQGSGGSGGTGSGGTGSGGSGTSMITSALHRA ADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVK LAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADE 50 KESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPEL GGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESL AQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRERFTPPQALRAQGLLKE AEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSK LDGFLAAKDLMAQGIKI MBD domain (SETB2) (SEQ ID NO: 25): GTLNLKGENPLQLPIKCHFQRRHAKTNSHSSALHVSYKTPCGRSLRNVEEVFRYLLETECNFLFTDNFSFNTYVQ 22

[0023] Example 5 – Adaptor sequencesBelow are examples of adaptor sequences, i.e. including sequencing linkers and, in some cases, barcodes, as disclosed. Barcode sequence is marked in bold. Tn5_MeA_P5_noBCD (SEQ ID NO: 26).5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 10 Tn5_MeA_P5_bcdA (SEQ ID NO: 27).5'-TCGTCGGCAGCGTCTCCACGC TATAGCCT GCGATCGAGGACGGCAGATGTGTATAAGAGACAGTn5_MeA_P5_bcdB (SEQ ID NO: 28)5'-TCGTCGGCAGCGTCTCCACGC ATAGAGGC GCGATCGAGGACGGCAGATGTGTATAAGAGACAGTn5_MeA_P5_bcdC (SEQ ID NO: 29)5'-TCGTCGGCAGCGTCTCCACGC CCTATCCT GCGATCGAGGACGGCAGATGTGTATAAGAGACAG20 Tn5ME-B (SEQ ID NO: 30):5′- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′Tn5MErev (SEQ ID NO: 31).5'-[phos]CTGTCTCTTATACACATCT-3' See detailed adaptor annealing and Tn5 loading protocol here. 30 Example 6 – Library structureFinal library structure of single-cell DNA methyla^on (single modality) using droplet-based pla^orm such 10x chromium (fig 5a) 23

[0024] Library sequencing (both read 1 and read 2 contain useful informa^on about DNA methyla^on) 1. Add (fig 5b) Nextera Read 1 primer to sequence thefirst read (bo^om strand as template, 50cycles) Illumina Nextera Read 1 primer: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' 2. Add (fig 5c) sample index sequencing primer (index 1) to sequence index (bo^om strand astemplate, 8 cycles) Sample index sequencing primer (index1): 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 10 3. Cluster regenera^on (fig 5d), add cell barcode sequencing primer (index2) to sequence thesecond index (i5) (top strand as template, 16 cycles, this is cell barcode) Cell barcode sequencing primer (index2): 5'- CTGTCTCTTATACACATCTGACGCTGCCGACGA -3' 4. Add (fig 5e) Nextera Read 2 primer to sequence the second read (top strand as template, 50cycles, DNA methyla^on read): Illumina Nextera Read 2 primer: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' 20 Final library structure of DNA methyla^on in bulk (fig 5f) There is no 16-nt single-cell barcode in bulk, index2 can be sequenced for 8 cycles in step 3. All the primers and other sequencing cycles are the same as in the single cell. Final library structure of single-cell DNA methyla^on and histone modifica^ons (mul^-modal) using droplet-based pla^orm such 10x chromium (fig 5g) 24

[0025] Library sequencing (both read 1 and read 2 contain useful informa^on about DNA methyla^on and histone modifica^on) 1. customer Read 1 primer to sequence thefirst read (bo^om strand as template, 36 cycles)Customer primer R1: 5'- GCGATCGAGGACGGCAGATGTGTATAAGAGACAG -3'2. Add sample index sequencing primer (index 1) to sequence index (bo^om strand as template,8 cycles) 10 Nextera primer I1: 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 3. Cluster regenera^on, add customer index2 primer to sequence the second index (i5) (topstrand as template, 48 cycles, this is cell barcode) Customer primer I2: 5'- CTGTCTCTTATACACATCTGCCGTCCTCGATCGC -3'4. Add Nextera Read 2 primer to sequence the second read (top strand as template, 36 cycles,DNA methyla^on read): Nextera primer R2: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'20 Final library structure of DNA methyla^on and histone modifica^ons (mul^-modal) in bulk (fig 5 h) 25

[0026] Sequencing primers in bulk are the same as in the single cells. Sequencing cycles can change to Read1: 50 cycles, Read2: 50 cycles, Index1: 8 cycle, and Index2: 8 cycle. References Yong, WS., Hsu, FM. & Chen, PY. Profiling genome-wide DNA methylation.^Epigenetics & Chromatin^9, 26 (2016). https: / / doi.org / 10.1186 / s13072-016-0075-3 Olova, N., Krueger, F., Andrews, S.^et al.^Comparison of whole-genome bisulfite sequencing library10 preparation strategies identifies sources of biases affecting DNA methylation data.^Genome Biol^19, 33 (2018). https: / / doi.org / 10.1186 / s13059-018-1408-2 Methyl-CpG-binding domain proteins: readers of the epigenome, 30 Apr 2015 https: / / doi.org / 10.2217 / epi.15.39 Bartosovic, M., Kabbe, M. & Castelo-Branco, G. Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues.^Nat Biotechnol^39, 825–835 (2021). https: / / doi.org / 10.1038 / s41587-021-00869-9 20 David Serre, Byron H. Lee, Angela H. Ting, MBD-isolated Genome Sequencing provides a high- throughput and comprehensive survey of DNA methylation in the human genome,^Nucleic Acids Research, Volume 38, Issue 2, 1 January 2010, Pages 391–399,^https: / / Vitak, S., Torkenczy, K., Rosenkrantz, J.^et al.^Sequencing thousands of single-cell genomes with combinatorial indexing.^Nat Methods^14, 302–308 (2017). https: / / doi.org / 10.1038 / nmeth.4154 The person skilled in the art realizes that the present disclosure is not limited to the preferred embodiments described above. The person skilled in the art further realizes that modifica^ons and 30 varia^ons are possible within the scope of the appended claims. For example, alterna^ve designs and of the method and kit depending on context may be contemplated, as well as alterna^ve ingredients and components, as long as the overall effects are achieved. Addi^onally, varia^ons to the disclosed embodiments can be understood and effected by the skilled person in prac^cing the claimed disclosure, from a study of the drawings, the disclosure, and the appended claims. 26

Claims

CLAIMS:

1. A method of profiling covalent DNA modifica^on(s) at a cellular or nuclei level comprising thesteps of: (a) providing a sample solu^on, comprising cell(s) or isolated nuclei comprising a nucleuscomprising genomic DNA, said genomic DNA comprising or is expected to comprisecovalent DNA modifica^on(s); (b) adding a buffer comprising a nucleosome deple^on agent to expose the genomic DNA;(c) adding a fusion protein comprising a DNA modifica^on binding domain and a nucleasepart;10 (d) incuba^ng the fusion protein together with the exposed genomic DNA under condi^onsallowing the fusion protein to bind to the genomic DNA; (e) washing the solu^on of step (d) with buffer to remove any excess fusion protein;(f) incuba^ng the solu^on of step (e) under condi^ons allowing the fusion protein to cut thegenomic DNA in proximity of the DNA modifica^on(s); (g) determining the sequence of the fusion protein incubated DNA of step (e) by high-throughput sequencing, thereby iden^fying the posi^on(s) of the DNA modifica^on(s) ofthe genomic DNA that have been sequenced.

2. The method of claim 1, wherein the fusion protein has the ability to tagment the modifiedbases of the genomic DNA to be profiled.20 3. The method of claim 2, wherein the nuclease part is a transposase (Tn) part which insertssequencing linkers and a detectable barcode into the genomic DNA in proximity to themodified bases.

4. The method of claim 3, wherein the inserted barcode uniquely iden^fies the DNAmodifica^on or modality.

5. The method of any one of the preceding claims, wherein the covalent DNA modifica^on to beprofiled is chosen from methyla^on, preferably 5-methylcytosine (5mC).

6. The method of any one of the preceding claims, wherein the nucleosome deple^on agent ischosen from lithium assisted nucleosome deple^on (LAND) or alterna^ve detergents, such as SDS, preferably LAND.30 7. The method of any one of the preceding claims, wherein the DNA modifica^on bindingdomain of the fusion protein is methyla^on binding domain (MBD), chosen from MBD1, MBD2, MBD3, MBD4, MBD5, MBD6, MBD7, MBD8, MBD9, MBD10, MBD11, MeCP2, BAZ2A, BAZ2B, SETB1 and SETB2, preferably MBD1 or MBD2.

8. The method of any one of claims 1 or 5-7, wherein the nuclease part of the fusion protein is aMicrococcal nuclease (MNase) or DNase I.

9. The method of any one of the preceding claims, wherein the nuclease part is a transposasepart (Tn), chosen from any transposase (Tn), preferably Tn5 or Tn7.

10. The method of any one of claims 4 to 9, wherein the means for detec^on of the nucleasepart comprises a unique barcode.40 11. The method of claim 10, wherein the barcode is a transposase-specific DNA barcode.

12. The method of any one of the preceding claims, for prepara^on of a sequencing librarycomprising profiling informa^on of covalent DNA modifica^ons, such as DNA methyla^on.

13. The method of any of the preceding claims, wherein the method is for mul^modal profiling.2714. The method according to claim 13, wherein the mul^modal profiling comprises profiling ofDNA methyla^on together with open chroma^n (ATAC-seq) or histone modifica^ons.

15. The method of any one of the preceding claims, suitable for use in applica^ons related tosingle-cell resolu^on, such as on 10X Genomics, or single-cell indexing methods, such as combinatorial indexing.

16. The method of any one of the preceding claims, suitable for use in applica^ons related tospa^al resolu^on, such as DBiT-sequencing or Visium, wherein mul^ple cells or regions of interest within a ^ssue are profiled in parallel.

17. A kit for use in DNA methyla^on profiling at single-cell or single-nuclei level of genomic DNA,10 comprising (i) reagents for use in lithium assisted nucleosome deple^on (LAND) and (ii) aMBD-nuclease fusion protein, comprising a DNA methyla^on binding domain and a nucleasepart.

18. The kit according to claim 17, wherein the MBD-nuclease fusion protein is chosen fromMBD1-MNase, MBD2-MNase, MBD1-DNase I, MBD2-DNase I, MBD1-Tn5, MBD1-Tn7, MBD2- Tn5 or MBD2-Tn7, for recogni^on of m5C methylated DNA.

19. The kit according to any one of claims 17 or 18, wherein the nuclease part comprises atransposase part, wherein the transposase part comprises sequencing linkers and means for detec^on, wherein the means for detec^on comprises unique barcodes.

20. The kit according to any one of claims 17 to 19, comprising:20 (i) a DEFND buffer for use in lithium assisted nucleosome deple^on, comprising NIBbuffer and lithium diiodosalicylate; (ii) a binding buffer, for use in incuba^on of MBD-nuclease fusion protein together witha nucleosome-depleted nuclei; (iii) an MBD-nuclease fusion protein;(iv) op^onally a wash buffer;(v) a tagmenta^on buffer;(vi) op^onally reagents for construc^on of sequencing library; and(vii) instruc^ons for use.

21. The kit according to any one of claims 17 to 20, suitable for use in mul^modal profiling.30 comprising profiling of DNA methyla^on together with open chroma^n (ATAC-seq) or histone modifica^ons. 28

Images