MNase-seq

Micrococcal nuclease (MNase) was first discovered in S. aureus in 1956,{{cite journal | vauthors = Cunningham L, Catlin BW, De Garilhe MP |title=A deoxyribonuclease of Micrococcus pyogenes |journal=Journal of the American Chemical Society |date=September 1956 |volume=78 |issue=18 |pages=4642–4645 |doi=10.1021/ja01599a031|bibcode=1956JAChS..78.4642C }} protein crystallized in 1966,{{cite journal | vauthors = Cotton FA, Hazen EE, Richardson DC | title = Crystalline extracellular nuclease of Staphylococcus aureus | journal = The Journal of Biological Chemistry | volume = 241 | issue = 19 | pages = 4389–90 | date = October 1966 | doi = 10.1016/S0021-9258(18)99732-2 | pmid = 5922963 | doi-access = free }} and characterized in 1967.{{cite journal | vauthors = Heins JN, Suriano JR, Taniuchi H, Anfinsen CB | title = Characterization of a nuclease produced by Staphylococcus aureus | journal = The Journal of Biological Chemistry | volume = 242 | issue = 5 | pages = 1016–20 | date = March 1967 | doi = 10.1016/S0021-9258(18)96225-3 | pmid = 6020427 | doi-access = free }} MNase digestion of chromatin was key to early studies of chromatin structure; being used to determine that each nucleosomal unit of chromatin was composed of approximately 200bp of DNA.{{cite journal | vauthors = Noll M | title = Subunit structure of chromatin | journal = Nature | volume = 251 | issue = 5472 | pages = 249–51 | date = September 1974 | pmid = 4422492 | doi = 10.1038/251249a0 | bibcode = 1974Natur.251..249N | s2cid = 637383 }} This, alongside Olins’ and Olins’ “beads on a string” model,{{cite journal | vauthors = Olins AL, Olins DE | title = Spheroid chromatin units (v bodies) | journal = Science | volume = 183 | issue = 4122 | pages = 330–2 | date = January 1974 | pmid = 4128918 | doi = 10.1126/science.183.4122.330 | bibcode = 1974Sci...183..330O | s2cid = 83480762 }} confirmed Kornberg’s ideas regarding the basic chromatin structure.{{cite journal | vauthors = Kornberg RD | title = Chromatin structure: a repeating unit of histones and DNA | journal = Science | volume = 184 | issue = 4139 | pages = 868–71 | date = May 1974 | pmid = 4825889 | doi = 10.1126/science.184.4139.868 | bibcode = 1974Sci...184..868K }} Upon additional studies, it was found that MNase could not degrade histone-bound DNA shorter than ~140bp and that DNase I and II could degrade the bound DNA to as low as 10bp.{{cite journal | vauthors = Keichline LD, Villee CA, Wassarman PM | title = Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases | journal = Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis | volume = 425 | issue = 1 | pages = 84–94 | date = February 1976 | pmid = 1247619 | doi = 10.1016/0005-2787(76)90218-5 }}{{cite journal|vauthors=Duerksen JD, Connor KW|date=April 1978|title=Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases|journal=Molecular and Cellular Biochemistry|volume=19|issue=2|pages=93–112|doi=10.1007/bf00232599|pmid=206820|s2cid=9230112}} This ultimately elucidated that ~146bp of DNA wrap around the nucleosome core,{{cite journal | vauthors = Kornberg RD, Lorch Y | title = Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome | journal = Cell | volume = 98 | issue = 3 | pages = 285–94 | date = August 1999 | pmid = 10458604 | doi = 10.1016/s0092-8674(00)81958-3 | s2cid = 14039910 | doi-access = free }} ~50bp linker DNA connect each nucleosome,{{cite journal | vauthors = Whitlock JP, Simpson RT | title = Removal of histone H1 exposes a fifty base pair DNA segment between nucleosomes | journal = Biochemistry | volume = 15 | issue = 15 | pages = 3307–14 | date = July 1976 | pmid = 952859 | doi = 10.1021/bi00660a022 }} and that 10 continuous base-pairs of DNA tightly bind to the core of the nucleosome in intervals.

In addition to being used to study chromatin structure, micrococcal nuclease digestion had been used in oligonucleotide sequencing experiments since its characterization in 1967.{{cite journal | vauthors = Feldmann H | title = [Sequence analysis of oliogonucleotides by means of micrococcal nuclease] | journal = European Journal of Biochemistry | volume = 2 | issue = 1 | pages = 102–5 | date = July 1967 | pmid = 6079759 | doi = 10.1111/j.1432-1033.1967.tb00113.x | doi-access = free }} MNase digestion was additionally used in several studies to analyze chromatin-free sequences, such as yeast (Saccharomyces cerevisiae) mitochondrial DNA{{cite journal | vauthors = Prunell A, Bernardi G | title = The mitochondrial genome of wild-type yeast cells. IV. Genes and spacers | journal = Journal of Molecular Biology | volume = 86 | issue = 4 | pages = 825–41 | date = July 1974 | pmid = 4610147 | doi = 10.1016/0022-2836(74)90356-8 }} as well as bacteriophage DNA{{cite journal | vauthors = Barrell BG, Weith HL, Donelson JE, Robertson HD | title = Sequence analysis of the ribosome-protected bacteriophase phiX174 DNA fragment containing the gene G initiation site | journal = Journal of Molecular Biology | volume = 92 | issue = 3 | pages = 377–93 | date = March 1975 | pmid = 1095758 | doi = 10.1016/0022-2836(75)90287-9 }}{{cite journal|vauthors=Bambara R, Wu R|date=June 1975|title=DNA sequence analysis. Terminal sequences of bacteriophage phi80|journal=The Journal of Biological Chemistry|volume=250|issue=12|pages=4607–18|doi=10.1016/S0021-9258(19)41345-8 |pmid=166999|doi-access=free }} through its preferential digestion of adenine and thymine-rich regions.{{cite journal | vauthors = Wingert L, Von Hippel PH | title = The conformation dependent hydrolysis of DNA by micrococcal nuclease | journal = Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis | volume = 157 | issue = 1 | pages = 114–26 | date = March 1968 | pmid = 4296058 | doi = 10.1016/0005-2787(68)90270-0 }} In the early 1980s, MNase digestion was used to determine the nucleosomal phasing and associated DNA for chromosomes from mature SV40,{{cite journal | vauthors = Hiwasa T, Segawa M, Yamaguchi N, Oda K | title = Phasing of nucleosomes in SV40 chromatin reconstituted in vitro | journal = Journal of Biochemistry | volume = 89 | issue = 5 | pages = 1375–89 | date = May 1981 | pmid = 6168635 | doi = 10.1093/oxfordjournals.jbchem.a133329 }} fruit flies (Drosophila melanogaster),{{cite journal | vauthors = Samal B, Worcel A, Louis C, Schedl P | title = Chromatin structure of the histone genes of D. melanogaster | journal = Cell | volume = 23 | issue = 2 | pages = 401–9 | date = February 1981 | pmid = 6258802 | doi = 10.1016/0092-8674(81)90135-5 | s2cid = 42138156 }} yeast,{{cite journal | vauthors = Lohr DE | title = Detailed analysis of the nucleosomal organization of transcribed DNA in yeast chromatin | journal = Biochemistry | volume = 20 | issue = 21 | pages = 5966–72 | date = October 1981 | pmid = 6272832 | doi = 10.1021/bi00524a007 }} and monkeys,{{cite journal | vauthors = Musich PR, Brown FL, Maio JJ | title = Nucleosome phasing and micrococcal nuclease cleavage of African green monkey component alpha DNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 79 | issue = 1 | pages = 118–22 | date = January 1982 | pmid = 6275381 | pmc = 345673 | doi = 10.1073/pnas.79.1.118 | bibcode = 1982PNAS...79..118M | doi-access = free }} among others. The first study to use this digestion to study the relevance of chromatin accessibility to gene expression in humans was in 1985. In this study, nuclease was used to find the association of certain oncogenic sequences with chromatin and nuclear proteins.{{cite journal | vauthors = Kasid UN, Hough C, Thraves P, Dritschilo A, Smulson M | title = The association of human c-Ha-ras sequences with chromatin and nuclear proteins | journal = Biochemical and Biophysical Research Communications | volume = 128 | issue = 1 | pages = 226–32 | date = April 1985 | pmid = 3885946 | doi = 10.1016/0006-291x(85)91668-7 }} Studies utilizing MNase digestion to determine nucleosome positioning without sequencing or array information continued into the early 2000s.{{cite journal | vauthors = Goriely S, Demonté D, Nizet S, De Wit D, Willems F, Goldman M, Van Lint C | title = Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites | journal = Blood | volume = 101 | issue = 12 | pages = 4894–902 | date = June 2003 | pmid = 12576336 | doi = 10.1182/blood-2002-09-2851 | doi-access = }}

With the advent of whole genome sequencing in the late 1990s and early 2000s, it became possible to compare purified DNA sequences to the eukaryotic genomes of S. cerevisiae,{{cite journal | vauthors = Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG | display-authors = 6 | title = Life with 6000 genes | journal = Science | volume = 274 | issue = 5287 | pages = 546, 563–7 | date = October 1996 | pmid = 8849441 | doi = 10.1126/science.274.5287.546 | bibcode = 1996Sci...274..546G | s2cid = 16763139 }} Caenorhabditis elegans,{{cite journal | title = Genome sequence of the nematode C. elegans: a platform for investigating biology | journal = Science | volume = 282 | issue = 5396 | pages = 2012–8 | date = December 1998 | pmid = 9851916 | doi = 10.1126/science.282.5396.2012 | bibcode = 1998Sci...282.2012. | author1 = The C. Elegans Sequencing Consortium }} D. melanogaster,{{cite journal | vauthors = Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, etal | title = The genome sequence of Drosophila melanogaster | journal = Science | volume = 287 | issue = 5461 | pages = 2185–95 | date = March 2000 | pmid = 10731132 | doi = 10.1126/science.287.5461.2185 | bibcode = 2000Sci...287.2185. }} Arabidopsis thaliana,{{cite journal | title = Analysis of the genome sequence of the flowering plant Arabidopsis thaliana | journal = Nature | volume = 408 | issue = 6814 | pages = 796–815 | date = December 2000 | pmid = 11130711 | doi = 10.1038/35048692 | bibcode = 2000Natur.408..796T | collaboration = The Arabidopsis Genome Initiative | doi-access = free | last1 = The Arabidopsis Genome Initiative }} Mus musculus,{{cite journal | vauthors = Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, etal | collaboration = Mouse Genome Sequencing Consortium | title = Initial sequencing and comparative analysis of the mouse genome | journal = Nature | volume = 420 | issue = 6915 | pages = 520–62 | date = December 2002 | pmid = 12466850 | doi = 10.1038/nature01262 | bibcode = 2002Natur.420..520W | doi-access = free }} and Homo sapiens.{{cite journal | title = Finishing the euchromatic sequence of the human genome | journal = Nature | volume = 431 | issue = 7011 | pages = 931–45 | date = October 2004 | pmid = 15496913 | doi = 10.1038/nature03001 | bibcode = 2004Natur.431..931H | author1 = International Human Genome Sequencing Consortium | doi-access = free }} MNase digestion was first applied to genome-wide nucleosome occupancy studies in S. cerevisiae{{cite journal | vauthors = Bernstein BE, Liu CL, Humphrey EL, Perlstein EO, Schreiber SL | title = Global nucleosome occupancy in yeast | journal = Genome Biology | volume = 5 | issue = 9 | pages = R62 | date = August 2004 | pmid = 15345046 | pmc = 522869 | doi = 10.1186/gb-2004-5-9-r62 | doi-access = free }} accompanied by analyses through microarrays to determine which DNA regions were enriched with MNase-resistant nucleosomes. MNase-based microarray analyses were often utilized at genome-wide scales for yeast{{cite journal | vauthors = Yuan GC, Liu YJ, Dion MF, Slack MD, Wu LF, Altschuler SJ, Rando OJ | title = Genome-scale identification of nucleosome positions in S. cerevisiae | journal = Science | volume = 309 | issue = 5734 | pages = 626–630 | date = July 2005 | pmid = 15961632 | doi = 10.1126/science.1112178 | bibcode = 2005Sci...309..626Y | s2cid = 43625066 | doi-access = free }}{{cite journal|vauthors=Lee W, Tillo D, Bray N, Morse RH, Davis RW, Hughes TR, Nislow C|date=October 2007|title=A high-resolution atlas of nucleosome occupancy in yeast|journal=Nature Genetics|volume=39|issue=10|pages=1235–44|doi=10.1038/ng2117|pmid=17873876|s2cid=12816925}} and in limited genomic regions in humans{{cite journal | vauthors = Ozsolak F, Song JS, Liu XS, Fisher DE | title = High-throughput mapping of the chromatin structure of human promoters | journal = Nature Biotechnology | volume = 25 | issue = 2 | pages = 244–8 | date = February 2007 | pmid = 17220878 | doi = 10.1038/nbt1279 | s2cid = 365969 }}{{cite journal|display-authors=6|vauthors=Dennis JH, Fan HY, Reynolds SM, Yuan G, Meldrim JC, Richter DJ, Peterson DG, Rando OJ, Noble WS, Kingston RE|date=June 2007|title=Independent and complementary methods for large-scale structural analysis of mammalian chromatin|journal=Genome Research|volume=17|issue=6|pages=928–39|doi=10.1101/gr.5636607|pmc=1891351|pmid=17568008}} to determine nucleosome positioning, which could be used as an inference for transcriptional inactivation.

In 2006, Next-Generation sequencing was first coupled with MNase digestion to explore nucleosome positioning and DNA sequence preferences in C. elegans,. This was the first example of MNase-seq in any organism.

It was not until 2008, around the time Next-Generation sequencing was becoming more widely available, when MNase digestion was combined with high-throughput sequencing, namely Solexa/Illumina sequencing, to study nucleosomal positioning at a genome-wide scale in humans. A year later, the terms “MNase-Seq” and “MNase-ChIP”, for micrococcal nuclease digestion with chromatin immunoprecipitation, were finally coined. Since its initial application in 2006, MNase-seq has been utilized to deep sequence DNA associated with nucleosome occupancy and epigenomics across eukaryotes. As of February 2020, MNase-seq is still applied to assay accessibility in chromatin.{{cite journal|vauthors=Zhao H, Zhang W, Zhang T, Lin Y, Hu Y, Fang C, Jiang J|date=February 2020|title=Genome-wide MNase hypersensitivity assay unveils distinct classes of open chromatin associated with H3K27me3 and DNA methylation in Arabidopsis thaliana|journal=Genome Biology|volume=21|issue=1|pages=24|doi=10.1186/s13059-020-1927-5|pmc=6996174|pmid=32014062 |doi-access=free }}

Chromatin is dynamic and the positioning of nucleosomes on DNA changes through the activity of various transcription factors and remodeling complexes, approximately reflecting transcriptional activity at these sites. DNA wrapped around nucleosomes are generally inaccessible to transcription factors.{{cite journal | vauthors = Hargreaves DC, Crabtree GR | title = ATP-dependent chromatin remodeling: genetics, genomics and mechanisms | journal = Cell Research | volume = 21 | issue = 3 | pages = 396–420 | date = March 2011 | pmid = 21358755 | pmc = 3110148 | doi = 10.1038/cr.2011.32 }} Hence, MNase-seq can be used to indirectly determine which regions of DNA are transcriptionally inaccessible by directly determining which regions are bound to nucleosomes.

In a typical MNase-seq experiment, eukaryotic cell nuclei are first isolated from a tissue of interest. Then, MNase-seq uses the endo-exonuclease micrococcal nuclease to bind and cleave protein-unbound regions of DNA of eukaryotic chromatin, first cleaving and resecting one strand, then cleaving the antiparallel strand as well. The chromatin can be optionally crosslinked with formaldehyde.{{cite journal | vauthors = Mieczkowski J, Cook A, Bowman SK, Mueller B, Alver BH, Kundu S, Deaton AM, Urban JA, Larschan E, Park PJ, Kingston RE, Tolstorukov MY | display-authors = 6 | title = MNase titration reveals differences between nucleosome occupancy and chromatin accessibility | journal = Nature Communications | volume = 7 | pages = 11485 | date = May 2016 | pmid = 27151365 | pmc = 4859066 | doi = 10.1038/ncomms11485 | bibcode = 2016NatCo...711485M }} MNase requires Ca²⁺ as a cofactor, typically with a final concentration of 1mM. If a region of DNA is bound by the nucleosome core (i.e. histones) or other chromatin-bound proteins (e.g. transcription factors), then MNase is unable to bind and cleave the DNA. Nucleosomes or the DNA-protein complexes can be purified from the sample and the bound DNA can be subsequently purified via gel electrophoresis and extraction. The purified DNA is typically ~150bp, if purified from nucleosomes, or shorter, if from another protein (e.g. transcription factors).{{cite journal | vauthors = Hainer SJ, Fazzio TG | title = Regulation of Nucleosome Architecture and Factor Binding Revealed by Nuclease Footprinting of the ESC Genome | journal = Cell Reports | volume = 13 | issue = 1 | pages = 61–69 | date = October 2015 | pmid = 26411677 | pmc = 4598306 | doi = 10.1016/j.celrep.2015.08.071 }} This makes short-read, high-throughput sequencing ideal for MNase-seq as reads for these technologies are highly accurate but can only cover a couple hundred continuous base-pairs in length.{{cite journal | vauthors = Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L, Law M | display-authors = 6 | title = Comparison of next-generation sequencing systems | journal = Journal of Biomedicine & Biotechnology | volume = 2012 | pages = 251364 | date = January 2012 | pmid = 22829749 | pmc = 3398667 | doi = 10.1155/2012/251364 | doi-access = free }} Once sequenced, the reads can be aligned to a reference genome to determine which DNA regions are bound by nucleosomes or proteins of interest, with tools such as Bowtie. The positioning of nucleosomes elucidated, through MNase-seq, can then be used to predict genomic expression{{cite journal | vauthors = Henikoff S | title = Nucleosome destabilization in the epigenetic regulation of gene expression | journal = Nature Reviews. Genetics | volume = 9 | issue = 1 | pages = 15–26 | date = January 2008 | pmid = 18059368 | doi = 10.1038/nrg2206 | s2cid = 24413271 }} and regulation{{cite journal | vauthors = Ercan S, Carrozza MJ, Workman JL | title = Global nucleosome distribution and the regulation of transcription in yeast | journal = Genome Biology | volume = 5 | issue = 10 | pages = 243 | date = September 2004 | pmid = 15461807 | pmc = 545588 | doi = 10.1186/gb-2004-5-10-243 | doi-access = free }} at the time of digestion.

File:MNASE application.png

Recently, MNase-seq has also been implemented in determining where transcription factors bind on the DNA.{{cite journal | vauthors = Gutin J, Sadeh R, Bodenheimer N, Joseph-Strauss D, Klein-Brill A, Alajem A, Ram O, Friedman N | display-authors = 6 | title = Fine-Resolution Mapping of TF Binding and Chromatin Interactions | journal = Cell Reports | volume = 22 | issue = 10 | pages = 2797–2807 | date = March 2018 | pmid = 29514105 | pmc = 5863041 | doi = 10.1016/j.celrep.2018.02.052 }}{{cite journal|vauthors=Skene PJ, Henikoff S|date=June 2015|title=A simple method for generating high-resolution maps of genome-wide protein binding|journal=eLife|volume=4|pages=e09225|doi=10.7554/eLife.09225|pmc=4480131|pmid=26079792 |doi-access=free }} Classical ChIP-seq displays issues with resolution quality, stringency in experimental protocol, and DNA fragmentation. Classical ChIP-seq typically uses sonication to fragment chromatin, which biases heterochromatic regions due to the condensed and tight binding of chromatin regions to each other. Unlike histones, transcription factors only transiently bind DNA. Other methods, such as sonication in ChIP-seq, requiring the use of increased temperatures and detergents, can lead to the loss of the factor. CUT&RUN sequencing is a novel form of an MNase-based immunoprecipitation. Briefly, it uses an MNase tagged with an antibody to specifically bind DNA-bound proteins that present the epitope recognized by that antibody. Digestion then specifically occurs at regions surrounding that transcription factor, allowing for this complex to diffuse out of the nucleus and be obtained without having to worry about significant background nor the complications of sonication. The use of this technique does not require high temperatures or high concentrations of detergent. Furthermore, MNase improves chromatin digestion due to its exonuclease and endonuclease activity. Cells are lysed in an SDS/Triton X-100 solution. Then, the MNase-antibody complex is added. And finally, the protein-DNA complex can be isolated, with the DNA being subsequently purified and sequenced. The resulting soluble extract contains a 25-fold enrichment in fragments under 50bp. This increased enrichment results in cost-effective high-resolution data.

Single-cell micrococcal nuclease sequencing (scMNase-seq) is a novel technique that is used to analyze nucleosome positioning and to infer chromatin accessibility with the use of only a single-cell input. First, cells are sorted into single aliquots using fluorescence-activated cell sorting (FACS). The cells are then lysed and digested with micrococcal nuclease. The isolated DNA is subjected to PCR amplification and then the desired sequence is isolated and analyzed. The use of MNase in single-cell assays results in increased detection of regions such as DNase I hypersensitive sites as well as transcription factor binding sites.

File:Sequencing table.png

MNase-seq is one of four major methods (DNase-seq, MNase-seq, FAIRE-seq, and ATAC-seq) for more direct determination of chromatin accessibility and the subsequent consequences for gene expression.{{cite journal | vauthors = Tsompana M, Buck MJ | title = Chromatin accessibility: a window into the genome | journal = Epigenetics & Chromatin | volume = 7 | issue = 1 | pages = 33 | date = November 2014 | pmid = 25473421 | pmc = 4253006 | doi = 10.1186/1756-8935-7-33 | doi-access = free }} All four techniques are contrasted with ChIP-seq, which relies on the inference that certain marks on histone tails are indicative of gene activation or repression,{{cite journal | vauthors = Park PJ | title = ChIP-seq: advantages and challenges of a maturing technology | journal = Nature Reviews. Genetics | volume = 10 | issue = 10 | pages = 669–80 | date = October 2009 | pmid = 19736561 | pmc = 3191340 | doi = 10.1038/nrg2641 }} not directly assessing nucleosome positioning, but instead being valuable for the assessment of histone modifier enzymatic function.

===DNase-seq===

As with MNase-seq, DNase-seq was developed by combining an existing DNA endonuclease with Next-Generation sequencing technology to assay chromatin accessibility.{{cite journal | vauthors = Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE | display-authors = 6 | title = High-resolution mapping and characterization of open chromatin across the genome | journal = Cell | volume = 132 | issue = 2 | pages = 311–22 | date = January 2008 | pmid = 18243105 | pmc = 2669738 | doi = 10.1016/j.cell.2007.12.014 }} Both techniques have been used across several eukaryotes to ascertain information on nucleosome positioning in the respective organisms and both rely on the same principle of digesting open DNA to isolate ~140bp bands of DNA from nucleosomes{{cite journal | vauthors = He HH, Meyer CA, Hu SS, Chen MW, Zang C, Liu Y, Rao PK, Fei T, Xu H, Long H, Liu XS, Brown M | display-authors = 6 | title = Refined DNase-seq protocol and data analysis reveals intrinsic bias in transcription factor footprint identification | journal = Nature Methods | volume = 11 | issue = 1 | pages = 73–78 | date = January 2014 | pmid = 24317252 | pmc = 4018771 | doi = 10.1038/nmeth.2762 }} or shorter bands if ascertaining transcription factor information. Both techniques have recently been optimized for single-cell sequencing, which corrects for one of the major disadvantages of both techniques; that being the requirement for high cell input.{{cite journal | vauthors = Cooper J, Ding Y, Song J, Zhao K | title = Genome-wide mapping of DNase I hypersensitive sites in rare cell populations using single-cell DNase sequencing | journal = Nature Protocols | volume = 12 | issue = 11 | pages = 2342–2354 | date = November 2017 | pmid = 29022941 | doi = 10.1038/nprot.2017.099 | s2cid = 7993995 | pmc = 11005227 }}{{cite journal | vauthors = Lai B, Gao W, Cui K, Xie W, Tang Q, Jin W, Hu G, Ni B, Zhao K | display-authors = 6 | title = Principles of nucleosome organization revealed by single-cell micrococcal nuclease sequencing | journal = Nature | volume = 562 | issue = 7726 | pages = 281–285 | date = October 2018 | pmid = 30258225 | doi = 10.1038/s41586-018-0567-3 | bibcode = 2018Natur.562..281L | s2cid = 52841785 | pmc = 8353605 }}

At sufficient concentrations, DNase I is capable of digesting nucleosome-bound DNA to 10bp, whereas micrococcal nuclease cannot. Additionally, DNase-seq is used to identify DHSs, which are regions of DNA that are hypersensitive to DNase treatment and are often indicative of regulatory regions (e.g. promoters or enhancers).{{cite journal | vauthors = Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, Sheffield NC, Stergachis AB, Wang H, Vernot B, Garg K, John S, Sandstrom R, Bates D, Boatman L, Canfield TK, Diegel M, Dunn D, Ebersol AK, Frum T, Giste E, Johnson AK, Johnson EM, Kutyavin T, Lajoie B, Lee BK, Lee K, London D, Lotakis D, Neph S, Neri F, Nguyen ED, Qu H, Reynolds AP, Roach V, Safi A, Sanchez ME, Sanyal A, Shafer A, Simon JM, Song L, Vong S, Weaver M, Yan Y, Zhang Z, Zhang Z, Lenhard B, Tewari M, Dorschner MO, Hansen RS, Navas PA, Stamatoyannopoulos G, Iyer VR, Lieb JD, Sunyaev SR, Akey JM, Sabo PJ, Kaul R, Furey TS, Dekker J, Crawford GE, Stamatoyannopoulos JA | display-authors = 6 | title = The accessible chromatin landscape of the human genome | journal = Nature | volume = 489 | issue = 7414 | pages = 75–82 | date = September 2012 | pmid = 22955617 | pmc = 2828505 | doi = 10.1038/nature11232 | bibcode = 2012Natur.489...75T }} An equivalent effect is not found with MNase. As a result of this distinction, DNase-seq is primarily utilized to directly identify regulatory regions, whereas MNase-seq is used to identify transcription factor and nucleosomal occupancy to indirectly infer effects on gene expression.

FAIRE-seq differs more from MNase-seq than does DNase-seq. FAIRE-seq was developed in 2007 and combined with Next-Generation sequencing three years later to study DHSs.{{cite journal | vauthors = Gaulton KJ, Nammo T, Pasquali L, Simon JM, Giresi PG, Fogarty MP, Panhuis TM, Mieczkowski P, Secchi A, Bosco D, Berney T, Montanya E, Mohlke KL, Lieb JD, Ferrer J | display-authors = 6 | title = A map of open chromatin in human pancreatic islets | journal = Nature Genetics | volume = 42 | issue = 3 | pages = 255–9 | date = March 2010 | pmid = 20118932 | pmc = 2828505 | doi = 10.1038/ng.530 }} FAIRE-seq relies on the use of formaldehyde to crosslink target proteins with DNA and then subsequent sonication and phenol-chloroform extraction to separate non-crosslinked DNA and crosslinked DNA. The non-crosslinked DNA is sequenced and analyzed, allowing for direct observation of open chromatin.{{cite journal | vauthors = Simon JM, Giresi PG, Davis IJ, Lieb JD | title = Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA | journal = Nature Protocols | volume = 7 | issue = 2 | pages = 256–67 | date = January 2012 | pmid = 22262007 | pmc = 3784247 | doi = 10.1038/nprot.2011.444 }}

MNase-seq does not measure chromatin accessibility as directly as FAIRE-seq. However, unlike FAIRE-seq, it does not necessarily require crosslinking, nor does it rely on sonication, but it may require phenol and chloroform extraction. Two major disadvantages of FAIRE-seq, relative to the other three classes, are the minimum required input of 100,000 cells and the reliance on crosslinking. Crosslinking may bind other chromatin-bound proteins that transiently interact with DNA, hence limiting the amount of non-crosslinked DNA that can be recovered and assayed from the aqueous phase. Thus, the overall resolution obtained from FAIRE-seq can be relatively lower than that of DNase-seq or MNase-seq and with the 100,000 cell requirement, the single-cell equivalents of DNase-seq or MNase-seq make them far more appealing alternatives.

ATAC-seq is the most recently developed class of chromatin accessibility assays. ATAC-seq uses a hyperactive transposase to insert transposable markers with specific adapters, capable of binding primers for sequencing, into open regions of chromatin. PCR can then be used to amplify sequences adjacent to the inserted transposons, allowing for determination of open chromatin sequences without causing a shift in chromatin structure. ATAC-seq has been proven effective in humans, amongst other eukaryotes, including in frozen samples.{{cite journal | vauthors = Corces MR, Buenrostro JD, Wu B, Greenside PG, Chan SM, Koenig JL, Snyder MP, Pritchard JK, Kundaje A, Greenleaf WJ, Majeti R, Chang HY | display-authors = 6 | title = Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution | journal = Nature Genetics | volume = 48 | issue = 10 | pages = 1193–203 | date = October 2016 | pmid = 27526324 | pmc = 5042844 | doi = 10.1038/ng.3646 }} As with DNase-seq and MNase-seq, a successful single-cell version of ATAC-seq has also been developed.{{cite journal | vauthors = Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ | display-authors = 6 | title = Single-cell chromatin accessibility reveals principles of regulatory variation | journal = Nature | volume = 523 | issue = 7561 | pages = 486–90 | date = July 2015 | pmid = 26083756 | pmc = 4685948 | doi = 10.1038/nature14590 | bibcode = 2015Natur.523..486B }}

ATAC-seq has several advantages over MNase-seq in assessing chromatin accessibility. ATAC-seq does not rely on the variable digestion of the micrococcal nuclease, nor crosslinking or phenol-chloroform extraction. It generally maintains chromatin structure, so results from ATAC-seq can be used to directly assess chromatin accessibility, rather than indirectly via MNase-seq. ATAC-seq can also be completed within a few hours, whereas the other three techniques typically require overnight incubation periods. The two major disadvantages to ATAC-seq, in comparison to MNase-seq, are the requirement for higher sequencing coverage and the prevalence of mitochondrial contamination due to non-specific insertion of DNA into both mitochondrial DNA and nuclear DNA. Despite these minor disadvantages, use of ATAC-seq over the alternatives is becoming more prevalent.

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Category:Molecular biology techniques