The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2, 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol. The reaction was stopped by adding half volume of deproteinization solution (20 mM Tris–HCl (pH 8.0), 20 mM EDTA, 0.5 mg/ml proteinase K (Roche) and 0.1% sodium dodecyl sulphate). The DNA fragments were purified by using Wizard SV Gel and PCR Clean-Up System (Promega). For the MNase digestion experiments with nuc19 and its mutants (Supplementary Table S1 ), each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above. The DNA fragments were extracted by phenol/chloroform/isoamyl alcohol and precipitated by ethanol. For the MNase digestion experiments with free DNAs, DNA mixture (94 nM) was incubated with 0.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as nucleosome digestion experiments. The DNA fragments were purified by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation.
Mnase
Manufactured by Takara Bio
Sourced in Japan
MNase is a type of enzyme that is commonly used in molecular biology and genomics research. It is a micrococcal nuclease, which is an enzyme that cleaves DNA and chromatin at specific sites. The primary function of MNase is to digest the DNA and chromatin between nucleosomes, allowing researchers to study the structure and organization of chromatin in cells.
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Lab products found in correlation
Proteinase k, by Roche (7 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Truseq dna lt sample prep kit, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Nebnext dna library prep master mix set, by Illumina (2 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Zymolyase, by Seikagaku (1 mentions)
Anti human mpo antibody, by Agilent Technologies (1 mentions)
Pazpc, by Cayman Chemical (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Sonifier sfx550, by Emerson (1 mentions)
Anti p300, by Active Motif (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Acetonitrile, by Beijing Chemical Works (1 mentions)
Sodium acetate, by Beijing Chemical Works (1 mentions)
Alkaline phosphatase, by Takara Bio (1 mentions)
34 protocols using mnase
1
Nucleosome Digestion by MNase
2
MNase Assays and Sequencing of Plant Nuclei
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The MNase assays were carried out as described previously (50 (link)) with minor modifications. Approximately 400 milligrams of seedlings were used per sample. Additionally, equal amounts of spike-in nuclei extracted from Saccharomyces cerevisiae were added to each sample before MNase treatment, which worked as the internal control among samples (51 (link)). For MNase treatment, the prepared nuclei were resuspended in prewarmed MNase digestion buffer, followed by the addition of 8 units of MNase (Cat. 2910A, TaKaRa) and incubation (15 min at 37°C with periodic agitation). Finally, the mono-nucleosomal DNA was isolated from 2% agarose gels and quantified by the Qubit dsDNA HS assay kit (Cat. Q32851, ThermoFisher SCIENTIFIC).
For MNase-qPCR, the nucleosome occupancy for a specific region was determined as the percentage of input-MNase-digested DNA, which was then normalized to the spike-in-control yeast NUC6. Primers used for qPCR are listed inSupplementary Dataset S1 . The locations of primers on each gene used for qPCR are shown in Supplementary Figure S1 . For MNase-seq, DNA libraries were generated following the published protocol (42 (link)).
For MNase-qPCR, the nucleosome occupancy for a specific region was determined as the percentage of input-MNase-digested DNA, which was then normalized to the spike-in-control yeast NUC6. Primers used for qPCR are listed in
3
MNase Accessibility Assay in Drosophila S2 Cells
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MNase accessibility assays were performed on equal amounts of collected dsRNA-treated S2 cells (described above). Cells were incubated for 10 min on ice with buffer A (15 mM Tris, pH 7.4; 60 mM KCl; 15 mM NaCl; 5 mM MgCl2; 300 mM sucrose; and 0.1% IGEPAL) and treated to 10 strokes using a Dounce homogenizer. Lysate was centrifuged and washed once with buffer A without detergent. Nuclei were then resuspended in MNase buffer (15 mM Tris, pH 7.4; 60 mM KCl; 15 mM NaCl; 3 mM CaCl2; and 200 mM sucrose) and digested at 37°C with 1 U MNase (#2910A; Takara). The reaction was stopped by adding 0.15 volumes of Stop solution (4% SDS and 100 mM EDTA). RNA and proteins were digested with 70 μg of RNase A for 1 h at 37°C followed by 70 μg of freshly made proteinase K for 2 h at 55°C. Digested DNA was purified with phenol chloroform extraction followed by ethanol precipitation. Finally, DNA was resuspended in TE (10 mM Tris, pH 8.0; and 1 mM EDTA) and analyzed on a 1.7% agarose gel stained with ethidium bromide.
4
Chromatin Isolation and MNase Digestion
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2 g of above ground tissue was harvested without crosslinking and nuclei and chromatin were isolated as previously described (Chodavarapu et al., 2010 (link)) with minor changes. The nuclear pellet was washed twice with HBB buffer. The isolated chromatin was digested with a final concentration of 0.2–0.5 units/μl MNase (Takara, Tokyo, Japan) for 3 min in digestion buffer at 37°C. Subsequent steps were performed as previously described (Chodavarapu et al., 2010 (link)). Relative nucleosome occupancy was analyzed by tiled oligo qPCR. Percent input enrichment for each primer pair was extrapolated using a dilution series of undigested genomic DNA (Gévry et al., 2009 (link)). Fold enrichment of nucleosome bound DNA was calculated by normalizing percent input of each primer pair over that of the gypsy-like retrotransposon (At4g07700). MNase in protoplasts was performed as in intact tissues with some modification. 2 × 106 cells were harvest by centrifugation at 11,800 rpm for 2 min, followed by resuspension in 500 µl lysis buffer by vortexing. After centrifugation at 7300 rpm for 5 min, the nuclear fraction was resuspended in HBC buffer (Chodavarapu et al., 2010 (link)). The chromatin was digested with a final concentration of 0.02 units/μl MNase (Takara). For primer sequences see Supplemental file 1 .
5
PIP Derivatives Nucleosome Isolation
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After PIP derivatives incubation, micrococcal nuclease (MNase) buffer [3] (10 mM Tris-HCl (pH 7.4), 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine and 0.1x protease inhibitor cocktail) pre-washed nuclei was subjected to MNase (Takara, Japan) digestion at 37 o C for 30 min. Sonication shearing may affect the non-covalently bound PIP, so we used MNase digestion. To avoid protein hindrance during affinity purification, the MNase digested nucleosomes were treated with proteinase K.
6
Chromatin Fractionation and Protein Extraction
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HeLa or HEK293T cells were synchronized by thymidine block and release in medium containing 100 ng/ml nocodazole. Mitotic cells were collected (1–2×107 cells/reaction) and washed by PBS once. Cell pellets were re-suspended in CSK1 buffer (10 mM PIPES, pH 6.8, 10% glycerol, 3 mM MgCl2, 100 mM NaCl, 1 mM DTT, 0.25 mM PMSF, 0.3% triton X-100) for 30 min on ice. Soluble fraction was collected as chromatin-unbound fraction or cytoplasmic fraction. Insoluble chromatin pellets were collected by centrifuging at 16,100 g for 10 min and washed again with CSK1 buffer. The chromatin pellets were then re-suspended in CSK2 buffer (10 mM PIPES, pH 6.8, 10% glycerol, 3 mM MgCl2, 250 mM NaCl, 2.5 mM CaCl2, 1 mM DTT, 0.25 mM PMSF) containing 20 units MNase (Takara) and incubated at 37°C for 30 min. After centrifugation at 16,100 g for 10 min, supernatant was collected as chromatin-bound protein fraction. Note: All CSK buffers contained complete mini protease inhibitor cocktail (Roche) and phosphatase inhibitor PhosSTOP (Roche) and were freshly prepared.
7
ChIP-seq Protocol for Histone Modifications
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The chromatin immunoprecipitations and subsequent deep sequencing in Figures 5 and 6 were also performed according to previous reports (Shang et al., 2010 (link), 2013 (link)). The MNase (Takara, Kyoto, Japan)-digested chromatin of 1.5 × 109 cells was extracted with 0.5 M NaCl–containing buffer, and the extract was exposed for 4 h at 4°C to Sepharose-Protein G beads preincubated with rabbit anti–CENP-A or -H3K9me3 antibodies (MBL, Nagoya, Japan). Beads were washed, and the bound DNA was purified and applied to deep sequencing.
ChIP-seq libraries were constructed as described in the Illumina TruSeq DNA LT Sample Prep Kit protocols. Briefly, ∼50 ng of purified DNAs were end repaired, followed by the 3′ addition of a single adenosine nucleotide and ligation to universal library adapters. DNA libraries were prepared by eight cycles of PCR amplification. ChIP DNA libraries were sequenced using the Illumina HiSeq 2500. Sequencing of libraries was performed up to 2 × 151 cycles. Image analysis and base calling were performed with the standard Illumina pipeline version RTA1.17.21.3.
Sequencing data were mapped to chicken genome database galGAL4 (UCSC Genome Browser) with a BWA 0.6.2 mapping tool (Li and Durbin, 2009 (link)). A 4% mismatch was allowed for the mapping.Figures 5 and 6 represent raw sequence reads. Input data are also shown.
ChIP-seq libraries were constructed as described in the Illumina TruSeq DNA LT Sample Prep Kit protocols. Briefly, ∼50 ng of purified DNAs were end repaired, followed by the 3′ addition of a single adenosine nucleotide and ligation to universal library adapters. DNA libraries were prepared by eight cycles of PCR amplification. ChIP DNA libraries were sequenced using the Illumina HiSeq 2500. Sequencing of libraries was performed up to 2 × 151 cycles. Image analysis and base calling were performed with the standard Illumina pipeline version RTA1.17.21.3.
Sequencing data were mapped to chicken genome database galGAL4 (UCSC Genome Browser) with a BWA 0.6.2 mapping tool (Li and Durbin, 2009 (link)). A 4% mismatch was allowed for the mapping.
8
N. crassa Nucleosome Sequencing
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N. crassa cells were grown and digested with micrococcal nuclease as previously described (McKnight et al., 2021 (link)) with the following modifications. MNase (Takara) concentration was optimized for each strain to yield ~80%–90% mononucleosomes (20 units for N3752, N3753, N7966, N8018, N7990, N7992, N7988, and N7989; 40 units for N6877; 60 units for N4718; and 80 units for N4730, N6170, N6171, N6876, N8016, and N8017). All digestions were for 10 min at 37°C, RNase (40 µg) treatment was for 1.5 h at 42°C, and proteinase K (200 µg) treatment was for 1 hr at 65°C. About 10 µg of gel-purified mononucleosome DNA was prepared for high-throughput sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Sequencing was performed by the University of Oregon Genomics and Cell Characterization Core Facility.
9
Isolation and Purification of Mononucleosomal DNA from S. complicata
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Equal volumes of S. complicata culture and 2% formaldehyde were mixed and incubated for 10 min. Next, 5 mL of 1.25 M glycine was added to the resulting solution. S. complicata cells were collected, washed with 50 mM Tris-EDTA buffer (pH 8), and then suspended in Zymolyase buffer (1 M sorbitol, 10 mM DTT, and 50 mM Tris-HCl, pH 8.0). Zymolyase (Seikagaku corporation, Japan) (50 U) was added to the cell suspension, and the resulting solution was incubated at 37 °C for 1 h. Cells were collected by centrifugation and suspended in 2.5 mL of Zymolyase buffer, after which 1 U of MNase (Takara, Japan) was added. The resulting digestion solution was incubated at 37 °C for 30 min, and the reaction was stopped by adding sodium dodecyl sulfate to a final concentration of 1% and EDTA to a final concentration of 10 mM. Proteinase K solution (5 µL) was added to the solution, and the mixture was incubated at 56 °C for 1 h. DNA was phenol/chloroform-extracted, ethanol-precipitated, and treated with RNase (Nippon Gene, Japan). Nucleosomal DNA fragments were isolated via electrophoresis on 2% agarose gel. The mononucleosomal DNA band was excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Germany).
10
Nucleosome Array MNase Digestion
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The purified di- and tri-nucleosome arrays (200 ng of DNA) were treated with 0, 0.05, 0.1, or 0.2 unit of MNase (Takara), in 5 μl of 22 mM Tris-HCl buffer (pH 7.5), containing 5 mM NaCl, 1 mM CaCl2, 1.3 mM DTT, 2.5 μg/ml BSA, and 5% glycerol. After a 3 min incubation at room temperature, the reactions were stopped by the addition of 60 μl of proteinase K solution (20 mM Tris-HCl (pH 7.5), 20 mM EDTA, 0.25% SDS, and 0.5 mg/ml proteinase K), and further incubated for 10 min. The DNA was then extracted by phenol/chloroform, and was precipitated with ethanol. The samples were fractionated by 6% PAGE in 0.2× TBE buffer.
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