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Mnase

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MNase is a nuclease enzyme that specifically cleaves DNA between nucleosomes, allowing for the analysis of chromatin structure. It is a useful tool for studying the organization and dynamics of chromatin within the nucleus.

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Lab products found in correlation

Hiseq 2500, by Illumina (2 mentions) Proteinase inhibitor cocktail, by Roche (2 mentions) Pcr purification kit, by Thermo Fisher Scientific (1 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Mnase, by New England Biolabs (1 mentions) Precr repair mix, by New England Biolabs (1 mentions) Superdex 200 10 30 gl, by GE Healthcare (1 mentions) Ecori, by New England Biolabs (1 mentions) Xbai, by New England Biolabs (1 mentions) Ls6500, by Beckman Coulter (1 mentions) Mini beadbeater 24, by Biospec (1 mentions) Genome analyzer iix, by Illumina (1 mentions) Truseq sample prep kit, by Illumina (1 mentions) Protein g agarose, by Merck Group (1 mentions) Mnase, by Merck Group (1 mentions) Zymolyase 100t, by Seikagaku (1 mentions) Dna library prep kit, by Illumina (1 mentions) Hiseq 2500 sequencing system, by Illumina (1 mentions) Dynabeads protein a, by Thermo Fisher Scientific (1 mentions) E gel system, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Micrococcal nuclease (MNase; (8 citations)

37 protocols using mnase

1

Mapping Nucleosome Positioning via MNase Digestion

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iSLK.219 nuclei were digested for 5 min at 37°C with a titration of MNase: 4 units/mL, 2 units/mL, and 1 unit/mL of MNase (Worthington Biochemical Corp.) in MNase cleavage buffer (4 mM MgCl2, 5 mM KCl, 50 mM Tris-Cl (pH 7.4), 1 mM CaCl2, 12.5% glycerol). The MNase digestion reactions were stopped with 50 mM EDTA. Next, the protein-DNA crosslinks were reversed by treating the MNase-digested nuclei with 0.2 mg/mL proteinase K and 1% sodium dodecyl sulfate, and incubating overnight at 60°C.
The samples were then run and the nucleosomal ladder was separated on a 2% agarose gel. Following the separation of the DNA fragments, mononucleosomally-sized and subnucleosomal-sized fragments (<200 bp) were isolated from the agarose gel, and the DNA was purified by electroelution. Next, the mononucleosomal- and subnucleosomal-sized fragments for all MNase concentrations were combined for each respective sample. Following the combination of all fragments per sample, DNA was extracted with phenol-chloroform and precipitated with alcohol for 10 min at −20°C. The DNA was then pelleted by centrifugation at 3000g for 10 min at 4°C, and dissolved in TE (0.1 mM EDTA, 10 mM Tris-Cl at pH 8.0).
Sexton B.S., Druliner B.R., Vera D.L., Avey D., Zhu F, & Dennis J.H. (2016). Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response. Oncotarget, 7(6), 6460-6475.
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2

Nucleosome Preparation and Chromatin Profiling

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Mouse 3134 cells were cultured, treated with vehicle or 100 nM dexamethasone for 1 h prior to harvest, and nuclei were prepared as described (23 (link)), except for the following modifications. Prior to MNase digestion, nuclei were washed in Nuclei Suspension Buffer (25% glycerol, 5 mM Mg-acetate, 5 mM HEPES pH 7.3, 0.08 mM EDTA, 0.5 mM spermidine, 1 mM DTT) and resuspended in MNase Digestion Buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 0.5 mM spermidine) at 75 million nuclei/ml and 400 U/ml MNase (Worthington). Nuclei were digested in 400 ul aliquots for 4–7 min at 37°C with gentle agitation; the reaction was terminated with Stop Buffer (500 mM NaCl (140 mM final), 50 mM EDTA (14 mM final), 20 mM EGTA (5.6 mM final), 3.6% SDS (1% final)) to yield ∼90% mono-nucleosomes (Supplementary Figure S1). The digests were treated with RNase (Life Technologies) for 30 min, then proteinase K (Ambion) for another 30 min. Samples were extracted twice with phenol/chloroform and ethanol-precipitated. DNA pellets were washed twice with 70% ethanol, dried and dissolved in water. Nicks in MNase-digested DNA were repaired using the PreCR repair mix (NEB) and the DNA was purified using a PCR purification kit (Life Technologies).
Johnson T.A., Chereji R.V., Stavreva D.A., Morris S.A., Hager G.L, & Clark D.J. (2017). Conventional and pioneer modes of glucocorticoid receptor interaction with enhancer chromatin in vivo. Nucleic Acids Research, 46(1), 203-214.
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3

Histone Octamer Formation and Nucleosome Reconstitution

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Recombinant human core histones (H2A, H2B, H3, and H4) were prepared and mixed at a 1.2:1.2:1:1 ratio in denaturing buffer and subsequently dialyzed into high salt native buffer to promote protein refolding and histone octamer formation (39 (link),40 (link)). Histone octamers were resolved from (H3/H4)2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography (Superdex 200 10/30GL, GE Healthcare), and selected fractions visualized by SDS-PAGE. Equimolar amounts of 601 DNA sequence (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) were mixed in high salt buffer, then dialyzed against a salt gradient to reconstitute nucleosomes (41 (link)). Nucleosomes were than resolved by Native PAGE. MNase susceptibility assays were performed on nucleosomes made with WT, E76Q and E76K H2B mutants by mixing each nucleosome (0.4 pmol) with 9.6U of MNase (Worthington) and incubating for indicated amount of time. Reactions were stopped with a 20mM EGTA + 20mM EDTA solution and resolved by native PAGE. Relative amounts of nucleosomes at each time point were measured by densitometry and plotted.
Bennett R.L., Bele A., Small E.C., Will C.M., Nabet B., Oyer J.A., Huang X., Ghosh R.P., Grzybowski A.T., Yu T., Zhang Q., Riva A., Lele T.P., Schatz G.C., Kelleher N.L., Ruthenburg A.J., Liphardt J, & Licht J.D. (2019). A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver. Cancer discovery, 9(10), 1438-1451.
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4

Nucleosome Mapping in Archaea

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T. acidophilum, P. calidifontis, T. kodakarensis, and S. solfataricus genomic DNA was incubated with MNase (Worthington Biochemical, Lakewood, NJ, United States) at various concentrations (0.3, 1, 3, 10 and 30 U MNase/100 μl) for 20 min at 37°C. The digestion reaction was stopped by adding stop solution (100 mM EDTA, 100 mM EGTA, 2% SDS). Chromosomal fragments were then purified with phenol/chloroform extraction and ethanol precipitation. The DNA pellet was dissolved in TE buffer (10 mM tris pH 7.5, 1 mM EDTA), resolved with a 2.5% agarose gel, and visualized with ethidium bromide staining.
Maruyama H., Prieto E.I., Nambu T., Mashimo C., Kashiwagi K., Okinaga T., Atomi H, & Takeyasu K. (2020). Different Proteins Mediate Step-Wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis. Frontiers in Microbiology, 11, 1247.
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5

Mononucleosome Profiling by MNase-seq

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At each time point and biological replicate ∼2.5 X 106 nuclei were treated with light (20U MNase, Worthington Biochemical) and heavy MNase-digestion conditions (200U MNase, Worthington Biochemical), see average fragment size distribution in Fig. S1B. Chromatin at each time point was digested separately with the light and heavy concentrations of MNase for five minutes at 37° and stopped with EDTA. Decrosslinked, protease-digested DNA from MNase-digested nuclei was isolated via phenol-chloroform extraction and mononucleosome sized bands resolved with a 2% TBE agarose gel. The ∼150bp mononucleosomal band was excised for each time point and MNase concentration. Additionally, two untreated control samples were harvested and processed as described, and have been referenced here as untreated samples.
Cole L, & Dennis J. (2020). MNase Profiling of Promoter Chromatin in Salmonella typhimurium-Stimulated GM12878 Cells Reveals Dynamic and Response-Specific Nucleosome Architecture. G3: Genes|Genomes|Genetics, 10(7), 2171-2178.
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6

MNase Digestion and Restriction Enzyme Accessibility

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Micrococcal nuclease (MNase) digestion was performed as described (13 (link)). Nuclei isolated from ESCs were digested with 5 and 30 U of MNase (Worthington, Lakewood, NJ, USA, www.worthington-biochem.com) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight. The purified DNA was subjected to Southern blot analysis. Restriction enzyme accessibility assays were carried out as described (13 (link)). Isolated nuclei from ESCs were digested with 100 U of SapI, AlwI, FokI, XbaI, PstI, EcoRI or StuI (New England Biolabs, Ipswich, MA, USA, www.neb.com) for 30 min. The purified genomic DNA was re-digested with 100 U of AvaI (for Nanog gene) or HindIII (for Oct4 gene). The digested fragments were analyzed by Southern blot using 32P-labeled probe (Figure 5A). All probes were listed in Supplementary Table S1.
Wu C.Y., Feng X, & Wei L.N. (2014). Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140. Nucleic Acids Research, 42(7), 4306-4317.
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7

Measuring Nascent Chromatin Replication

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Chromatin was prelabeled with [14C]thymidine (0.5 pCi/ml) for 24 hours before siRNA transfection. Thirty hours after transfection, nascent chromatin was labeled with [3H]thymidine (25 nCi/ml). To compensate for lower replication rate in TLK2- and FLASH-depleted cells, labeling times were adjusted to obtain similar [3H]thymidine incorporation (10, 15, and 30 min for siRNA control, siTLK2, and siFLASH, respectively). Cells were lysed in hypotonic buffer [10 mM tris (pH 7.4), 2.5 mM MgCl2, and 0.5% NP-40, protease and phosphatase inhibitors], and nuclei were resuspended in digestion buffer [10 mM tris (pH 7.4), 10 mM NaCl, 5 mM MgCl2, and 2 mM CaCl2, protease and phosphatase inhibitors] and subjected to digestion with MNase (0.01 U/μl) (Worthington Biochemical Co.) at 37°C. Undigested chromatin was pelleted by centrifugation at 1500g for 2 min, and 14C and 3H activity in supernatant and undigested chromatin were measured with a liquid scintillation counter (LS 6500, Beckman Coulter). Readings were corrected for 14C bleed through into the 3H channel. Graphs show one representative experiment of three biological replicates.
Lee S.B., Segura-Bayona S., Villamor-Payà M., Saredi G., Todd M.A., Attolini C.S., Chang T.Y., Stracker T.H, & Groth A. (2018). Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors. Science Advances, 4(8), eaat4985.
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8

Chromatin Isolation and Mononucleosome Extraction

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Detailed conditions of cell culture, chromatin isolation, MNase digestion and DNA extraction have been previously described (Zhou and O'Shea, 2011 (link)). Briefly, 100 OD600 units of yeast cells (EY57; genotype K699, ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL+) growing in synthetic complete medium at 30°C with shaking were harvested at OD600 ~0.5 for each experiment. Cells were crosslinked with 1% formaldehyde for 15 min at room temperature and quenched with 125 mM glycine for 5 min. Collected cells were lysed mechanically with glass beads at 4°C with Mini-Beadbeater-24 (Biospec) in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% Sodium-Deoxycholate) and the chromatin pellet fraction was digested with either 0.5, 1, 2 or 4 U MNase (Worthington Biochemical) at 37°C for 30 min. DNA fragments corresponding to mono-nucleosomes were extracted from an agarose gel after electrophoretic separation.
Zhou X., Blocker A.W., Airoldi E.M, & O'Shea E.K. (2016). A computational approach to map nucleosome positions and alternative chromatin states with base pair resolution. eLife, 5, e16970.
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9

Ribosome Profiling: Monosome Isolation

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Ribosome profiling was performed as described [56 (link)]. Briefly, the pulverized cells were thawed and the soluble cytoplasmic fraction isolated by centrifugation of insoluble material. The clarified lysates were treated with micrococcal nuclease to degrade DNA and reduce polysomes to monosomes (MNase, Worthington Biochemical Corp). Monosomes were isolated by sucrose-gradient fractionation. mRNA was extracted from the monosome fraction by treatment with acid phenol and chloroform extraction, and isopropanol precipitation.
Shell S.S., Wang J., Lapierre P., Mir M., Chase M.R., Pyle M.M., Gawande R., Ahmad R., Sarracino D.A., Ioerger T.R., Fortune S.M., Derbyshire K.M., Wade J.T, & Gray T.A. (2015). Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape. PLoS Genetics, 11(11), e1005641.
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10

MNase-Digested Chromatin Profiling

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MNase-digested chromatin was prepared as described previously with some modifications [23 (link), 77 (link), 78 (link)]. After fixation, imaginal tissues were resuspended in 1 ml buffer A (300 mM sucrose, 2 mM Mg acetate, 3 mM CaCl2, 10 mM Tris pH 8, 0.1 % Triton X-100 and 0.5 mM DTT), for 60 larvae and homogenized in a Dounce homogenizer (tight pestle, Wheaton). Nuclei were collected by centrifugation at 4 °C, 720 g for 5 min, washed by buffer A and washed in buffer D (50 mM Tris–HCl pH 8, 25 % glycerol, 5 mM Mg acetate, 0.1 mM EDTA and 5 mM DTT). Nuclei were resuspended in buffer MN (15 mM Tris–HCl pH 8.5, 60 mM KCl, 15 mM NaCl, 0.5 mM DTT, 0.25 M sucrose and 3 mM CaCl2). Two hundred microliter of chromatin was digested with 20U of MNase (Worthington) for 3 h at 25 °C. Gel-purified DNA fragments ranging from 100 to 200 bp were then subjected to pair-end sequencing by an Illumina Genome Analyzer IIx.
Details of bioinformatic analyses are presented in Additional file 3.
Tsai S.Y., Chang Y.L., Swamy K.B., Chiang R.L, & Huang D.H. (2016). GAGA factor, a positive regulator of global gene expression, modulates transcriptional pausing and organization of upstream nucleosomes. Epigenetics & Chromatin, 9, 32.
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