Structured Review

Thermo Fisher mnase
<t>DKC1-associated</t> RNAs in recombinant DKC1 complexes are resistant to extensive <t>MNase</t> digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
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Images

1) Product Images from "The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells"

Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

Journal: eLife

doi: 10.7554/eLife.03573

DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
Figure Legend Snippet: DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

Techniques Used: Recombinant, Purification, Labeling, Stable Transfection

Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009
Figure Legend Snippet: Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

Techniques Used: Recombinant, Mutagenesis, Staining

MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011
Figure Legend Snippet: MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

Techniques Used: Activity Assay, In Vitro, Concentration Assay, Recombinant

2) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000639

Neutrophils release calprotectin by forming NETs. (A–F) Confocal images of human neutrophils without stimulation (A), after 0.5 h (B), 1 h (C), 2 h (D), 3 h (E) and 4 h (F) after activation. Samples were stained with antibodies specific for the calprotectin heteroduplex (red) and for MPO (green). DNA was stained with DRAQ5 (blue). Calprotectin localizes to the cytoplasm and partially to the nucleus (A, arrow). After stimulation for 0.5 h (B) the neutrophils flattened and formed numerous vacuoles. This reveals a granular staining for MPO and a more dispersed cytoplasmic staining for calprotectin. After stimulation for 1 h (C) the neutrophils round up slightly. The MPO and calprotectin stain partially overlap in the cytoplasm. After stimulation for 2 h (D), calprotectin, MPO and nuclear DNA start to colocalize in the decondensed nucleus (purple). After 3 h (E) and more so after 4 h (F) of stimulation, the cell membrane ruptures and calprotectin is released in NETs colocalizing with MPO and DNA. Scale bar = 10 µm; one experiment out of two is shown. (G–I) Subunits of calprotectin S100A8 and S100A9 are released after cell death during NET formation and not by degranulation. NET formation was induced with PMA and degranulation using formyl-met-leu-phe (f-MLP). (G) Neutrophil death was monitored by quantification of LDH activity in supernatants calculated as means±s.d. (n = 3). (H) Release of S100A8, S100A9, lactotransferrin (LTF) and myeloperoxidase (MPO) were analyzed by immunoblotting. one experiment out of two is shown. (I) Quantification of immunoblots using 2D densitometry analyzing S100A9 protein preparations from supernatants (lane 1), MNase-digested NETs (lane 2) and cell remnants indigestible for MNase (lane 3). Values were calculated as means±s.d. (n = 3) from one experiment out of two.
Figure Legend Snippet: Neutrophils release calprotectin by forming NETs. (A–F) Confocal images of human neutrophils without stimulation (A), after 0.5 h (B), 1 h (C), 2 h (D), 3 h (E) and 4 h (F) after activation. Samples were stained with antibodies specific for the calprotectin heteroduplex (red) and for MPO (green). DNA was stained with DRAQ5 (blue). Calprotectin localizes to the cytoplasm and partially to the nucleus (A, arrow). After stimulation for 0.5 h (B) the neutrophils flattened and formed numerous vacuoles. This reveals a granular staining for MPO and a more dispersed cytoplasmic staining for calprotectin. After stimulation for 1 h (C) the neutrophils round up slightly. The MPO and calprotectin stain partially overlap in the cytoplasm. After stimulation for 2 h (D), calprotectin, MPO and nuclear DNA start to colocalize in the decondensed nucleus (purple). After 3 h (E) and more so after 4 h (F) of stimulation, the cell membrane ruptures and calprotectin is released in NETs colocalizing with MPO and DNA. Scale bar = 10 µm; one experiment out of two is shown. (G–I) Subunits of calprotectin S100A8 and S100A9 are released after cell death during NET formation and not by degranulation. NET formation was induced with PMA and degranulation using formyl-met-leu-phe (f-MLP). (G) Neutrophil death was monitored by quantification of LDH activity in supernatants calculated as means±s.d. (n = 3). (H) Release of S100A8, S100A9, lactotransferrin (LTF) and myeloperoxidase (MPO) were analyzed by immunoblotting. one experiment out of two is shown. (I) Quantification of immunoblots using 2D densitometry analyzing S100A9 protein preparations from supernatants (lane 1), MNase-digested NETs (lane 2) and cell remnants indigestible for MNase (lane 3). Values were calculated as means±s.d. (n = 3) from one experiment out of two.

Techniques Used: Activation Assay, Staining, Activity Assay, Western Blot

3) Product Images from "Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns"

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.450

Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)
Figure Legend Snippet: Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)

Techniques Used: Genomic Sequencing, Polymerase Chain Reaction, Amplification, Fluorescence, Staining, Western Blot, Imaging, Transmission Electron Microscopy

4) Product Images from "Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster ▿"

Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01409-06

Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was
Figure Legend Snippet: Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was

Techniques Used: Isolation, Purification

5) Product Images from "CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe"

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe

Journal: The EMBO Journal

doi: 10.1038/emboj.2012.289

Hrp1 and Hrp3 efficiently space nucleosomes in vitro in an ATP-dependent manner. Phage λ DNA salt dialysis chromatin was incubated with Hrp1, Hrp3 and ATP as indicated above the lanes and digested with MNase. Wedges on top of the lanes correspond to increasing concentrations of 1, 2 and 4 U/ml. ‘M' denotes 100 bp marker (New England Biolabs).
Figure Legend Snippet: Hrp1 and Hrp3 efficiently space nucleosomes in vitro in an ATP-dependent manner. Phage λ DNA salt dialysis chromatin was incubated with Hrp1, Hrp3 and ATP as indicated above the lanes and digested with MNase. Wedges on top of the lanes correspond to increasing concentrations of 1, 2 and 4 U/ml. ‘M' denotes 100 bp marker (New England Biolabs).

Techniques Used: In Vitro, Incubation, Marker

Related Articles

Centrifugation:

Article Title: Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression
Article Snippet: Each sample was incubated with 200 units of MNase (Fermentas) at 37°C for 5 min, followed by the addition of 4 mM EDTA to halt MNase digestion. .. Following centrifugation at 1500 g for 5 min at 4°C, the supernatant (S1) was retained.

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: The nucleoplasmic fraction was separated by centrifugation at 14,000 rpm at 4 °C f or 10 min. Chromatin-bound proteins were released from the DNA by addition of 0.2 N HCl and incubation for 10 min on ice, followed by centrifugation at 14,000 rpm at 4 °C fo r 10 min and neutralization with 1M Tris-HCl. .. Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h.

Article Title: Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation
Article Snippet: For MNase digest, the nuclei concentration was adjusted to 20 A260 in nuclei buffer R (85 mM KCL, 5.5% sucrose, 10 mM Tris, pH 7.5, 1 mM CaCl2 , 1 mM MgCl2 , and 250 µM PMSF) and digested with 8 U MNase (Thermo Fisher Scientific) for 8 min at RT. .. Nuclear debris was removed through centrifugation, and the soluble chromatin was divided into two samples (one brought to 650 mM NaCl) before fractionation on sucrose step gradients.

Neutralization:

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: The nucleoplasmic fraction was separated by centrifugation at 14,000 rpm at 4 °C f or 10 min. Chromatin-bound proteins were released from the DNA by addition of 0.2 N HCl and incubation for 10 min on ice, followed by centrifugation at 14,000 rpm at 4 °C fo r 10 min and neutralization with 1M Tris-HCl. .. Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h.

Picogreen Assay:

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns
Article Snippet: Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification. .. Total DNA and released DNA were quantified using a PicoGreen dsDNA assay kit (Life Technologies, Seoul, Korea) according to the manufacturer's instructions.

Cell Fractionation:

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: Paragraph title: Immunoblotting, cellular fractionation and co-immunoprecipitation ... Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h.

Construct:

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe
Article Snippet: .. We constructed a pht1 Δ swr1 Δ double deletion mutant and measured genome-wide nucleosome occupancy by hybridizing MNase generated mononucleosomal DNA fragments to Affymetrix tiling arrays. ..

Electrophoresis:

Article Title: Different nucleosomal architectures at early and late replicating origins in Saccharomyces cerevisiae
Article Snippet: Digestion with MNase and indirect end-labelling analyses 2×109 cells were permeabilized as described above and spheroplasts were split into six fractions in NP-buffer and treated with increasing amounts of MNase (Fermentas) (0, 0.5, 1, 1.5, 2 and 3 units/ml) for 10 minutes at 37°C. .. For each sample, 2.5 μg of DNA were digested with HindIII and PvuII enzymes (Fermentas), separated by electrophoresis in a 1.5% agarose gel, blotted and hybridized to an specific end-terminal probe.

Incubation:

Article Title: Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression
Article Snippet: .. Each sample was incubated with 200 units of MNase (Fermentas) at 37°C for 5 min, followed by the addition of 4 mM EDTA to halt MNase digestion. .. Following centrifugation at 1500 g for 5 min at 4°C, the supernatant (S1) was retained.

Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
Article Snippet: .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction. .. The DNA fragments were then purified using a MinElute PCR purification kit (Qiagen) and analyzed on an Agilent 2100 Bioanalyzer as previously described .

Article Title: Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts
Article Snippet: The chromatin was digested by adding MNase (Thermo Scientific, 88216) for 20min at 37°C and MNase was inactivated by adding 20mM EGTA. .. DNA was purified by adding RNase A (Thermo Scientific, EN0531) and incubated for 30 min at 37°C and then with Proteinase K (Invitrogen, 25530049) for 2h.

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: The nucleoplasmic fraction was separated by centrifugation at 14,000 rpm at 4 °C f or 10 min. Chromatin-bound proteins were released from the DNA by addition of 0.2 N HCl and incubation for 10 min on ice, followed by centrifugation at 14,000 rpm at 4 °C fo r 10 min and neutralization with 1M Tris-HCl. .. Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h.

Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
Article Snippet: Peak fractions containing all four subunits of the DKC1 complex, as determined by western blotting, were pooled and incubated with anti-FLAG (M2) agarose (Sigma Aldrich) for 3–4 hr, washed at 0.5 M NaCl HEGN and re-equilibrated with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl, pH 7.9, 20 mM NaCl, 60 mM KCl, 2 mM CaCl2 , 0.01% NP-40, 10% glycerol). .. Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
Article Snippet: 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor. .. For each transfected sample, 260 ul of MNase:Lysis buffer was added and incubated for 15 minutes at 25 C, and 20 minutes at 37C.

Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster ▿
Article Snippet: .. For micrococcal nuclease (MNase) digestion, the nucleus solutions were made 3 mM CaCl2 and 0.5 units/μl MNase (Fermentas) and incubated at 24°C for the specified time. ..

Activity Assay:

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns
Article Snippet: .. Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification. .. Total genomic DNA from HeLa cells was extracted with 500 μ l DNazol (Molecular Research Center, Cincinnati, OH, USA).

Expressing:

Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr. .. For purification of bacterial DKC1 complexes, pST44 expression plasmids were transformed into BL21-Codon Plus RIPL competent cells (Agilent).

Genome Wide:

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe
Article Snippet: .. We constructed a pht1 Δ swr1 Δ double deletion mutant and measured genome-wide nucleosome occupancy by hybridizing MNase generated mononucleosomal DNA fragments to Affymetrix tiling arrays. ..

Modification:

Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
Article Snippet: Native ChIP Quantitative measurements of histone modification levels were preformed in parallel using native ChIP. .. 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor.

Western Blot:

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h. .. Proteins were eluted by addition of 2 × SDS sample buffer (0.125 M Tris-HCl [pH 6.8], 4% SDS, 20% glycerol and 0.01% bromophenol blue, 5% 2-β-mercaptoethanol, protease/phosphatase inhibitors) and incubation at 95°C for 5 min. Lysates of equal protein amount based on BCA assay were separated by SDS-PAGE and subjected to western blotting.

Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
Article Snippet: Peak fractions containing all four subunits of the DKC1 complex, as determined by western blotting, were pooled and incubated with anti-FLAG (M2) agarose (Sigma Aldrich) for 3–4 hr, washed at 0.5 M NaCl HEGN and re-equilibrated with micrococcal nuclease (MNase) digestion buffer (25 mM Tris–HCl, pH 7.9, 20 mM NaCl, 60 mM KCl, 2 mM CaCl2 , 0.01% NP-40, 10% glycerol). .. Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

Transformation Assay:

Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr. .. For purification of bacterial DKC1 complexes, pST44 expression plasmids were transformed into BL21-Codon Plus RIPL competent cells (Agilent).

Flow Cytometry:

Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
Article Snippet: Peak fractions were loaded immediately to a gravity column containing Heparin Sepharose 6 Fast Flow (GE Healthcare) pre-equilibrated with 0.3 M NaCl HEGN (25 mM HEPES, pH 7.9, 0.1 mM EDTA, 10% glycerol, 0.02% NP-40). .. Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

Southern Blot:

Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster ▿
Article Snippet: Paragraph title: Nuclease digestions and Southern blotting. ... For micrococcal nuclease (MNase) digestion, the nucleus solutions were made 3 mM CaCl2 and 0.5 units/μl MNase (Fermentas) and incubated at 24°C for the specified time.

Immunoprecipitation:

Article Title: ACF chromatin remodeling complex mediates stress–induced depressive–like behavior
Article Snippet: .. Tissue was lightly fixed to cross–link DNA (12 minutes with 1% formaldehyde, following by 5 minutes with glycine, and 8x cold PBS washes) with associated proteins, and the material was further sheared (for BAZ1A and SMARCA5, Bioruptor to obtain mostly 100–300 bp fragments) or MNase digested (for histone H3/nucleosome, nuclei were digested with 10 U/mL MNase at 37 °C for 10 min, reaction stopped with adding EDTA to 20 mM, > 80% mononucleosome obtained) and immunoprecipitated using sheep anti–rabbit magnetic beads (Invitrogen) conjugated to an antibody that specifically recognizes BAZ1A (Bethyl), SMARCA5, or H3 (Abcam). .. Immunoprecipitated DNA and total (input) genomic DNA were prepared for ChIP–seq using an Illumina kit according to the manufacturer’s instructions.

Protease Inhibitor:

Article Title: Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression
Article Snippet: Nuclei were then washed, and resuspended in 1 mL of MNase digestion buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2 , 0.1% NP40, 0.25M sucrose, 3mM CaCl2 , 10 mM N-ethylmaleimide, 1x protease inhibitor cocktail (Roche)). .. Each sample was incubated with 200 units of MNase (Fermentas) at 37°C for 5 min, followed by the addition of 4 mM EDTA to halt MNase digestion.

Article Title: Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts
Article Snippet: The cells were then lysed with lysis buffer (100mM Tris-HCl, 300mM NaCl, 2% Triton® X-100, 0.2%v sodium deoxycholate and 10mM Cacl2) supplemented with EDTA-free protease inhibitor (Roche, 11873580001) for 20min in Ice. .. The chromatin was digested by adding MNase (Thermo Scientific, 88216) for 20min at 37°C and MNase was inactivated by adding 20mM EGTA.

Cell Culture:

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns
Article Snippet: Quantification of released DNAs Cells were cultured in six-well plates for > 24 h to 80% confluency and treated with various cytotoxic reagents for the indicated time periods. .. Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification.

Generated:

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe
Article Snippet: .. We constructed a pht1 Δ swr1 Δ double deletion mutant and measured genome-wide nucleosome occupancy by hybridizing MNase generated mononucleosomal DNA fragments to Affymetrix tiling arrays. ..

Sequencing:

Article Title: ACF chromatin remodeling complex mediates stress–induced depressive–like behavior
Article Snippet: Paragraph title: ChIP, library preparation, and sequencing ... Tissue was lightly fixed to cross–link DNA (12 minutes with 1% formaldehyde, following by 5 minutes with glycine, and 8x cold PBS washes) with associated proteins, and the material was further sheared (for BAZ1A and SMARCA5, Bioruptor to obtain mostly 100–300 bp fragments) or MNase digested (for histone H3/nucleosome, nuclei were digested with 10 U/mL MNase at 37 °C for 10 min, reaction stopped with adding EDTA to 20 mM, > 80% mononucleosome obtained) and immunoprecipitated using sheep anti–rabbit magnetic beads (Invitrogen) conjugated to an antibody that specifically recognizes BAZ1A (Bethyl), SMARCA5, or H3 (Abcam).

Binding Assay:

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe
Article Snippet: We constructed a pht1 Δ swr1 Δ double deletion mutant and measured genome-wide nucleosome occupancy by hybridizing MNase generated mononucleosomal DNA fragments to Affymetrix tiling arrays. .. First, we distinguished targets from non-targets in terms of H2A.Z binding (ChIP-chip data of ( )) ( ).

Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
Article Snippet: Paragraph title: Nucleosome in vitro binding and MNase digestion assays ... Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

ChIP-chip:

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe
Article Snippet: We constructed a pht1 Δ swr1 Δ double deletion mutant and measured genome-wide nucleosome occupancy by hybridizing MNase generated mononucleosomal DNA fragments to Affymetrix tiling arrays. .. First, we distinguished targets from non-targets in terms of H2A.Z binding (ChIP-chip data of ( )) ( ).

ChIP-sequencing:

Article Title: ACF chromatin remodeling complex mediates stress–induced depressive–like behavior
Article Snippet: For each ChIP–seq replicate, bilateral 14–gauge NAc punches were pooled from 5–10 mice. .. Tissue was lightly fixed to cross–link DNA (12 minutes with 1% formaldehyde, following by 5 minutes with glycine, and 8x cold PBS washes) with associated proteins, and the material was further sheared (for BAZ1A and SMARCA5, Bioruptor to obtain mostly 100–300 bp fragments) or MNase digested (for histone H3/nucleosome, nuclei were digested with 10 U/mL MNase at 37 °C for 10 min, reaction stopped with adding EDTA to 20 mM, > 80% mononucleosome obtained) and immunoprecipitated using sheep anti–rabbit magnetic beads (Invitrogen) conjugated to an antibody that specifically recognizes BAZ1A (Bethyl), SMARCA5, or H3 (Abcam).

DNA Extraction:

Article Title: Restitution of gene expression and histone acetylation signatures altered by hepatitis B virus through antiviral microRNA-like molecules in nontransformed murine hepatocytes
Article Snippet: MNase digest time courses of each 1 × 106 cells were performed for 0, 1, 2, 4, 8, 16 or 20 minutes with 0.15 U/μL MNase (Thermo Fisher Scientific, Waltham, MA, USA). .. The reactions were stopped by directly mixing 50 μL phenol:chloroform:isoamylalcohol with each sample followed by DNA extraction.

Nucleic Acid Electrophoresis:

Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
Article Snippet: Recombinant protein VII-His was then combined with nucleosomes at various molar ratios, incubated at room temperature for 15 minutes, and analyzed by native gel electrophoresis. .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

Magnetic Beads:

Article Title: ACF chromatin remodeling complex mediates stress–induced depressive–like behavior
Article Snippet: .. Tissue was lightly fixed to cross–link DNA (12 minutes with 1% formaldehyde, following by 5 minutes with glycine, and 8x cold PBS washes) with associated proteins, and the material was further sheared (for BAZ1A and SMARCA5, Bioruptor to obtain mostly 100–300 bp fragments) or MNase digested (for histone H3/nucleosome, nuclei were digested with 10 U/mL MNase at 37 °C for 10 min, reaction stopped with adding EDTA to 20 mM, > 80% mononucleosome obtained) and immunoprecipitated using sheep anti–rabbit magnetic beads (Invitrogen) conjugated to an antibody that specifically recognizes BAZ1A (Bethyl), SMARCA5, or H3 (Abcam). .. Immunoprecipitated DNA and total (input) genomic DNA were prepared for ChIP–seq using an Illumina kit according to the manufacturer’s instructions.

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h. .. Nuclear lysate was incubated with Anti-Flag M2 Magnetic Beads (Sigma Aldrich) at 4°C for 1h, and beads were washed with wash buffer (20 mM HEPES [pH 8.0], 150 mM KCl and 0.1% NP-40) 4 times and with 50 mM HEPES (pH 8.0) once.

Mutagenesis:

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe
Article Snippet: .. We constructed a pht1 Δ swr1 Δ double deletion mutant and measured genome-wide nucleosome occupancy by hybridizing MNase generated mononucleosomal DNA fragments to Affymetrix tiling arrays. ..

Transfection:

Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
Article Snippet: 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor. .. For each transfected sample, 260 ul of MNase:Lysis buffer was added and incubated for 15 minutes at 25 C, and 20 minutes at 37C.

Electrophoretic Mobility Shift Assay:

Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
Article Snippet: Nucleosome in vitro binding and MNase digestion assays Gel shift and MNase digestion assays were carried out as previously described , , . .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

Mouse Assay:

Article Title: ACF chromatin remodeling complex mediates stress–induced depressive–like behavior
Article Snippet: For each ChIP–seq replicate, bilateral 14–gauge NAc punches were pooled from 5–10 mice. .. Tissue was lightly fixed to cross–link DNA (12 minutes with 1% formaldehyde, following by 5 minutes with glycine, and 8x cold PBS washes) with associated proteins, and the material was further sheared (for BAZ1A and SMARCA5, Bioruptor to obtain mostly 100–300 bp fragments) or MNase digested (for histone H3/nucleosome, nuclei were digested with 10 U/mL MNase at 37 °C for 10 min, reaction stopped with adding EDTA to 20 mM, > 80% mononucleosome obtained) and immunoprecipitated using sheep anti–rabbit magnetic beads (Invitrogen) conjugated to an antibody that specifically recognizes BAZ1A (Bethyl), SMARCA5, or H3 (Abcam).

Polymerase Chain Reaction:

Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
Article Snippet: Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction. .. The DNA fragments were then purified using a MinElute PCR purification kit (Qiagen) and analyzed on an Agilent 2100 Bioanalyzer as previously described .

Recombinant:

Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
Article Snippet: Recombinant protein VII-His was then combined with nucleosomes at various molar ratios, incubated at room temperature for 15 minutes, and analyzed by native gel electrophoresis. .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

Purification:

Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
Article Snippet: Briefly, nucleosomes were reconstituted by incubating purified recombinant histones with ‘601’ DNA of either 195 or 147bp over a series of dialysis. .. Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

Article Title: Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts
Article Snippet: The chromatin was digested by adding MNase (Thermo Scientific, 88216) for 20min at 37°C and MNase was inactivated by adding 20mM EGTA. .. DNA was purified by adding RNase A (Thermo Scientific, EN0531) and incubated for 30 min at 37°C and then with Proteinase K (Invitrogen, 25530049) for 2h.

Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells
Article Snippet: Paragraph title: Reconstitution and purification of the DKC1 complexes ... Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

Chromatin Immunoprecipitation:

Article Title: ACF chromatin remodeling complex mediates stress–induced depressive–like behavior
Article Snippet: Paragraph title: ChIP, library preparation, and sequencing ... Tissue was lightly fixed to cross–link DNA (12 minutes with 1% formaldehyde, following by 5 minutes with glycine, and 8x cold PBS washes) with associated proteins, and the material was further sheared (for BAZ1A and SMARCA5, Bioruptor to obtain mostly 100–300 bp fragments) or MNase digested (for histone H3/nucleosome, nuclei were digested with 10 U/mL MNase at 37 °C for 10 min, reaction stopped with adding EDTA to 20 mM, > 80% mononucleosome obtained) and immunoprecipitated using sheep anti–rabbit magnetic beads (Invitrogen) conjugated to an antibody that specifically recognizes BAZ1A (Bethyl), SMARCA5, or H3 (Abcam).

Article Title: Restitution of gene expression and histone acetylation signatures altered by hepatitis B virus through antiviral microRNA-like molecules in nontransformed murine hepatocytes
Article Snippet: MNase digest time courses of each 1 × 106 cells were performed for 0, 1, 2, 4, 8, 16 or 20 minutes with 0.15 U/μL MNase (Thermo Fisher Scientific, Waltham, MA, USA). .. From the aqueous phase DNA was precipitated with isopropanol, resolved in water and DNA fragments were analyzed on an Agilent Bioanalyzer 2100 using a DNA chip.

Article Title: Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts
Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) ... The chromatin was digested by adding MNase (Thermo Scientific, 88216) for 20min at 37°C and MNase was inactivated by adding 20mM EGTA.

Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
Article Snippet: Paragraph title: Native ChIP ... 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor.

SDS Page:

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h. .. Proteins were eluted by addition of 2 × SDS sample buffer (0.125 M Tris-HCl [pH 6.8], 4% SDS, 20% glycerol and 0.01% bromophenol blue, 5% 2-β-mercaptoethanol, protease/phosphatase inhibitors) and incubation at 95°C for 5 min. Lysates of equal protein amount based on BCA assay were separated by SDS-PAGE and subjected to western blotting.

Co-Immunoprecipitation Assay:

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: For co-IP assays, cells were trypsinized, PBS-washed and lysed in hypotonic buffer (20 mM HEPES [pH 8.0], 10 mM KCl, 2 mM MgCl2 , 10% glycerol, 320 mM sucrose, 0.5% NP-40 and protease inhibitors). .. Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h.

Selection:

Article Title: ACF chromatin remodeling complex mediates stress–induced depressive–like behavior
Article Snippet: Tissue was lightly fixed to cross–link DNA (12 minutes with 1% formaldehyde, following by 5 minutes with glycine, and 8x cold PBS washes) with associated proteins, and the material was further sheared (for BAZ1A and SMARCA5, Bioruptor to obtain mostly 100–300 bp fragments) or MNase digested (for histone H3/nucleosome, nuclei were digested with 10 U/mL MNase at 37 °C for 10 min, reaction stopped with adding EDTA to 20 mM, > 80% mononucleosome obtained) and immunoprecipitated using sheep anti–rabbit magnetic beads (Invitrogen) conjugated to an antibody that specifically recognizes BAZ1A (Bethyl), SMARCA5, or H3 (Abcam). .. Proprietary adapters were then ligated to the ends, followed by size selection on a 2% agarose gel.

Agarose Gel Electrophoresis:

Article Title: ACF chromatin remodeling complex mediates stress–induced depressive–like behavior
Article Snippet: Tissue was lightly fixed to cross–link DNA (12 minutes with 1% formaldehyde, following by 5 minutes with glycine, and 8x cold PBS washes) with associated proteins, and the material was further sheared (for BAZ1A and SMARCA5, Bioruptor to obtain mostly 100–300 bp fragments) or MNase digested (for histone H3/nucleosome, nuclei were digested with 10 U/mL MNase at 37 °C for 10 min, reaction stopped with adding EDTA to 20 mM, > 80% mononucleosome obtained) and immunoprecipitated using sheep anti–rabbit magnetic beads (Invitrogen) conjugated to an antibody that specifically recognizes BAZ1A (Bethyl), SMARCA5, or H3 (Abcam). .. Proprietary adapters were then ligated to the ends, followed by size selection on a 2% agarose gel.

Article Title: Different nucleosomal architectures at early and late replicating origins in Saccharomyces cerevisiae
Article Snippet: Digestion with MNase and indirect end-labelling analyses 2×109 cells were permeabilized as described above and spheroplasts were split into six fractions in NP-buffer and treated with increasing amounts of MNase (Fermentas) (0, 0.5, 1, 1.5, 2 and 3 units/ml) for 10 minutes at 37°C. .. For each sample, 2.5 μg of DNA were digested with HindIII and PvuII enzymes (Fermentas), separated by electrophoresis in a 1.5% agarose gel, blotted and hybridized to an specific end-terminal probe.

In Vitro:

Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals
Article Snippet: Paragraph title: Nucleosome in vitro binding and MNase digestion assays ... Complexes were also digested with MNase (Affymetrix) by addition of 1 unit per µg of DNA for 147bp nucleosome experiments and 0.1 unit per µg of DNA for 195bp nucleosome experiments, incubated at 22°C for varying amounts of time followed by the addition of EGTA and guanidine thiocyanate to stop the reaction.

Concentration Assay:

Article Title: Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation
Article Snippet: .. For MNase digest, the nuclei concentration was adjusted to 20 A260 in nuclei buffer R (85 mM KCL, 5.5% sucrose, 10 mM Tris, pH 7.5, 1 mM CaCl2 , 1 mM MgCl2 , and 250 µM PMSF) and digested with 8 U MNase (Thermo Fisher Scientific) for 8 min at RT. .. Digestion was stopped by addition of EDTA to 10 mM, the sample was spun down, and chromatin was released overnight at 4°C in TEEP20 (10 mM Tris, pH 8, 1 mM EDTA, 1 mM EGTA, 250 µM PMSF, and 20 mM NaCl) containing 300 ng/ml l -α-lysophosphatidylcholine (Sigma-Aldrich).

Fractionation:

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: For chromatin fractionation, cells were resuspended in 0.5 ml lysis buffer containing 10 mM HEPES (pH 7.4), 10 mM KCl, 0.5% NP-40 with phosphatase inhibitors (Calbiochem), and protease inhibitors and incubated on ice for 20 min. .. Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h.

Article Title: Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation
Article Snippet: For MNase digest, the nuclei concentration was adjusted to 20 A260 in nuclei buffer R (85 mM KCL, 5.5% sucrose, 10 mM Tris, pH 7.5, 1 mM CaCl2 , 1 mM MgCl2 , and 250 µM PMSF) and digested with 8 U MNase (Thermo Fisher Scientific) for 8 min at RT. .. Nuclear debris was removed through centrifugation, and the soluble chromatin was divided into two samples (one brought to 650 mM NaCl) before fractionation on sucrose step gradients.

Lysis:

Article Title: Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression
Article Snippet: Mixed cells were pelleted and nuclei were released by resuspending in ice cold lysis buffer (10mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2 , 0.1% NP40, 5 mM N-ethylmaleimide). .. Each sample was incubated with 200 units of MNase (Fermentas) at 37°C for 5 min, followed by the addition of 4 mM EDTA to halt MNase digestion.

Article Title: Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts
Article Snippet: The cells were then lysed with lysis buffer (100mM Tris-HCl, 300mM NaCl, 2% Triton® X-100, 0.2%v sodium deoxycholate and 10mM Cacl2) supplemented with EDTA-free protease inhibitor (Roche, 11873580001) for 20min in Ice. .. The chromatin was digested by adding MNase (Thermo Scientific, 88216) for 20min at 37°C and MNase was inactivated by adding 20mM EGTA.

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: The nuclear pellet was washed in lysis buffer, pelleted and resuspended in 250 μl low salt buffer, containing 10 mM Tris-HCl (pH 7.4), 0.2 mM MgCl2, 1% Triton X-100 with phosphatase inhibitors and protease inhibitors, and incubated on ice for 15 min. .. Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h.

Article Title: Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions
Article Snippet: .. 0.01 U of MNase (ThermoScientific) was added to 1 ml lysis buffer (50mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM CaCl2) with EDTA free proteinase inhibitor. .. For each transfected sample, 260 ul of MNase:Lysis buffer was added and incubated for 15 minutes at 25 C, and 20 minutes at 37C.

Variant Assay:

Article Title: CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe
Article Snippet: Paragraph title: Neither the histone variant H2A.Z nor the remodeling enzyme Swr1 has a major role in nucleosome positioning ... We constructed a pht1 Δ swr1 Δ double deletion mutant and measured genome-wide nucleosome occupancy by hybridizing MNase generated mononucleosomal DNA fragments to Affymetrix tiling arrays.

BIA-KA:

Article Title: Replication stress shapes a protective chromatin environment across fragile genomic regions
Article Snippet: Nuclei were collected and resuspended in MNase digestion buffer (20 mM HEPES [pH 8.0], 150 mM, KCl, 10% glycerol, 3 mM CaCl2 and protease inhibitors), and treated with MNase (Thermo Fisher Scientific) at 4 °C for 1h. .. Proteins were eluted by addition of 2 × SDS sample buffer (0.125 M Tris-HCl [pH 6.8], 4% SDS, 20% glycerol and 0.01% bromophenol blue, 5% 2-β-mercaptoethanol, protease/phosphatase inhibitors) and incubation at 95°C for 5 min. Lysates of equal protein amount based on BCA assay were separated by SDS-PAGE and subjected to western blotting.

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  • 90
    Thermo Fisher micrococcal nuclease mnase
    Micrococcal Nuclease Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/micrococcal nuclease mnase/product/Thermo Fisher
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    micrococcal nuclease mnase - by Bioz Stars, 2020-01
    90/100 stars
      Buy from Supplier

    95
    Thermo Fisher mnase
    <t>DKC1-associated</t> RNAs in recombinant DKC1 complexes are resistant to extensive <t>MNase</t> digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
    Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase/product/Thermo Fisher
    Average 95 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    mnase - by Bioz Stars, 2020-01
    95/100 stars
      Buy from Supplier

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    DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Recombinant, Purification, Labeling, Stable Transfection

    Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Recombinant, Mutagenesis, Staining

    MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Activity Assay, In Vitro, Concentration Assay, Recombinant