Structured Review

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CUT RUN has low background when performed on ice. During protocol optimization, we performed the cleavage reactions over a range of temperatures. ( A ) We initially used <t>37°C</t> as is often done for <t>MNase</t> reactions. Careful analysis of the data, however, showed that despite clearly mapping CTCF at its true sites with a low density genome-wide background, we also had a specific background at random DNase1 sites. We rationalized that specific background arose from the liberated chromatin complexes that are still bound by Protein A-MNase diffusing around the nucleus and cutting accessible regions of chromatin. ( B ) To test this hypothesis, after the CTCF antibody and Protein A-MNase had bound in situ, we disrupted the nuclear envelope with limited sonication to release the chromatin into the large reaction volume. When CUT RUN was performed under disrupted conditions, we no longer observed this specific background. ( C ) We therefore tried to limit the diffusion of these chromatin complexes by performing the cleavage reaction at room temperature. We observed that the signal-to-noise ratio started low, but increased over time and by 8 min the noise was indistinguishable from the signal. ( D ) However, by keeping the reaction on ice the signal-to-noise ratio was hi and independent of time. Therefore, by controlling the temperature for the cleavage reaction, we can robustly maintain a low background. DOI: http://dx.doi.org/10.7554/eLife.21856.018
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1) Product Images from "An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites"

Article Title: An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites

Journal: eLife

doi: 10.7554/eLife.21856

CUT RUN has low background when performed on ice. During protocol optimization, we performed the cleavage reactions over a range of temperatures. ( A ) We initially used 37°C as is often done for MNase reactions. Careful analysis of the data, however, showed that despite clearly mapping CTCF at its true sites with a low density genome-wide background, we also had a specific background at random DNase1 sites. We rationalized that specific background arose from the liberated chromatin complexes that are still bound by Protein A-MNase diffusing around the nucleus and cutting accessible regions of chromatin. ( B ) To test this hypothesis, after the CTCF antibody and Protein A-MNase had bound in situ, we disrupted the nuclear envelope with limited sonication to release the chromatin into the large reaction volume. When CUT RUN was performed under disrupted conditions, we no longer observed this specific background. ( C ) We therefore tried to limit the diffusion of these chromatin complexes by performing the cleavage reaction at room temperature. We observed that the signal-to-noise ratio started low, but increased over time and by 8 min the noise was indistinguishable from the signal. ( D ) However, by keeping the reaction on ice the signal-to-noise ratio was hi and independent of time. Therefore, by controlling the temperature for the cleavage reaction, we can robustly maintain a low background. DOI: http://dx.doi.org/10.7554/eLife.21856.018
Figure Legend Snippet: CUT RUN has low background when performed on ice. During protocol optimization, we performed the cleavage reactions over a range of temperatures. ( A ) We initially used 37°C as is often done for MNase reactions. Careful analysis of the data, however, showed that despite clearly mapping CTCF at its true sites with a low density genome-wide background, we also had a specific background at random DNase1 sites. We rationalized that specific background arose from the liberated chromatin complexes that are still bound by Protein A-MNase diffusing around the nucleus and cutting accessible regions of chromatin. ( B ) To test this hypothesis, after the CTCF antibody and Protein A-MNase had bound in situ, we disrupted the nuclear envelope with limited sonication to release the chromatin into the large reaction volume. When CUT RUN was performed under disrupted conditions, we no longer observed this specific background. ( C ) We therefore tried to limit the diffusion of these chromatin complexes by performing the cleavage reaction at room temperature. We observed that the signal-to-noise ratio started low, but increased over time and by 8 min the noise was indistinguishable from the signal. ( D ) However, by keeping the reaction on ice the signal-to-noise ratio was hi and independent of time. Therefore, by controlling the temperature for the cleavage reaction, we can robustly maintain a low background. DOI: http://dx.doi.org/10.7554/eLife.21856.018

Techniques Used: Genome Wide, In Situ, Sonication, Diffusion-based Assay

2) Product Images from "DNA sequence encoded repression of rRNA gene transcription in chromatin"

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq263

rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. Nucleosomal DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with MNase, and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].
Figure Legend Snippet: rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. Nucleosomal DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with MNase, and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].

Techniques Used: Clear Native PAGE, Incubation, Purification, Clone Assay, In Vivo, Sequencing

PK inhibits nucleosome remodelling at the rDNA promoter. ( A ) MNase digestion of chromatin assembled on the rDNA minigene (pMrWT-T). Chromatin reconstituted with the Drosophila extract was digested with MNase for 0.5–3 min (lanes 1–3) or for 0.5–6 min (lanes 4–8) in the presence of 600 μM PK. The nucleosomal ladder (1n-5n) and the DNA marker (M; 1kb ladder) are indicated. ( B ) Chromatin assembled on pMrWT-T was incubated in the absence or presence of TTF-I and partially digested with MNase. Purified DNA was digested with EcoRI, separated on an agarose gel and transferred onto a nylon membrane. Chromatin configuration around the TTF-I-binding site (T 0 ) was visualized by indirect end-labelling (lanes 1 and 2). Chromatin remodelling was monitored in the presence of K (lanes 4 and 5; 300 and 600 μM) or PK (lanes 6 and 7, 300 and 600 μM). The position of the TTF-I-binding site is indicated by Sal I digestion of the template DNA (lane 3). Open circles mark non-positioned nucleosomes, whereas the gray circles indicate positioned nucleosomes. The position of the TTF-I-binding site (gray box), MNase-protected DNA regions (black triangles) and MNase-sensitive regions (white triangles) are indicated. The strong band in lane 6 (marked with an asterisk) arises due to the relatively lower MNase digestion of this sample.
Figure Legend Snippet: PK inhibits nucleosome remodelling at the rDNA promoter. ( A ) MNase digestion of chromatin assembled on the rDNA minigene (pMrWT-T). Chromatin reconstituted with the Drosophila extract was digested with MNase for 0.5–3 min (lanes 1–3) or for 0.5–6 min (lanes 4–8) in the presence of 600 μM PK. The nucleosomal ladder (1n-5n) and the DNA marker (M; 1kb ladder) are indicated. ( B ) Chromatin assembled on pMrWT-T was incubated in the absence or presence of TTF-I and partially digested with MNase. Purified DNA was digested with EcoRI, separated on an agarose gel and transferred onto a nylon membrane. Chromatin configuration around the TTF-I-binding site (T 0 ) was visualized by indirect end-labelling (lanes 1 and 2). Chromatin remodelling was monitored in the presence of K (lanes 4 and 5; 300 and 600 μM) or PK (lanes 6 and 7, 300 and 600 μM). The position of the TTF-I-binding site is indicated by Sal I digestion of the template DNA (lane 3). Open circles mark non-positioned nucleosomes, whereas the gray circles indicate positioned nucleosomes. The position of the TTF-I-binding site (gray box), MNase-protected DNA regions (black triangles) and MNase-sensitive regions (white triangles) are indicated. The strong band in lane 6 (marked with an asterisk) arises due to the relatively lower MNase digestion of this sample.

Techniques Used: Marker, Incubation, Purification, Agarose Gel Electrophoresis, Binding Assay

TTF-I-dependent chromatin dynamics at the rRNA gene promoter. Reconstituted nucleosomal arrays were incubated for 90 min with the TxE, TTF-I, K (600 μM) or PK (600 μM) as indicated. Nucleosome positions at the rDNA promoter were mapped by partial MNase digestion and primer extension of the purified DNA. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager. The graph shows the positions (relative to the transcription start site, +1; site is marked by boxes) and relative intensities of the MNase cleavage sites corresponding to the 3′ boundaries of positioned nucleosomes. Boxes highlight the MNase cleavage sites around the transcription start site, correlating with the nucleosome position nucAct . The position of the oligonucleotide used for primer extension and the major MNase-sensitive sites on the rDNA are indicated. The scan of the DNA marker (10-bp ladder) is shown below the graphs.
Figure Legend Snippet: TTF-I-dependent chromatin dynamics at the rRNA gene promoter. Reconstituted nucleosomal arrays were incubated for 90 min with the TxE, TTF-I, K (600 μM) or PK (600 μM) as indicated. Nucleosome positions at the rDNA promoter were mapped by partial MNase digestion and primer extension of the purified DNA. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager. The graph shows the positions (relative to the transcription start site, +1; site is marked by boxes) and relative intensities of the MNase cleavage sites corresponding to the 3′ boundaries of positioned nucleosomes. Boxes highlight the MNase cleavage sites around the transcription start site, correlating with the nucleosome position nucAct . The position of the oligonucleotide used for primer extension and the major MNase-sensitive sites on the rDNA are indicated. The scan of the DNA marker (10-bp ladder) is shown below the graphs.

Techniques Used: Incubation, Purification, Sequencing, Marker

3) Product Images from "Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription"

Article Title: Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription

Journal:

doi: 10.1101/gad.244434.114

TF binding at both category I and category II promoters overlaps with unusually MNase-labile chromatin. ( A ) Chromatin was underdigested or overdigested with MNase and sequenced (see the Materials and Methods). The average relative signal (a proxy for
Figure Legend Snippet: TF binding at both category I and category II promoters overlaps with unusually MNase-labile chromatin. ( A ) Chromatin was underdigested or overdigested with MNase and sequenced (see the Materials and Methods). The average relative signal (a proxy for

Techniques Used: Binding Assay

4) Product Images from "Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules"

Article Title: Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

Journal: Genes & Development

doi: 10.1101/gad.466908

Hmo1 is a structural constituent of actively transcribed 35S rDNA chromatin. ( A ) Hmo1 remains associated with rDNA in the absence of Pol I. ChEC was performed with nuclei isolated from yeast strains cultured for 2 h at 37°C, carrying either a wild-type ( RRN3 ) or a temperature-sensitive ( rrn3 -ts) allele of the essential, Pol I-specific initiation factor Rrn3, and expressing the MNase fusion protein indicated at the top of the panel. Isolated nuclei were incubated in calcium-containing buffer at 30°C for the minutes depicted above each lane. DNA was isolated, digested with XcmI, and analyzed in a Southern blot as described in the legend for A cartoon of the genomic region is depicted on the right as described in the legend for An arrow points to the uncut XcmI fragment of the genomic DNA region. Two fragments of unknown origin are labeled by asterisks. ( B ) S-band chromatin can be established in the absence of Hmo1. Psoralen cross-link analyses were performed with nuclei isolated from yeast strains carrying either a wild-type ( HMO1 , y881) or a complete deletion (Δ, y1159) of the HMO1 gene. DNA was isolated, digested with EcoRI, and analyzed in a Southern blot as described in the legend for A profile analysis after quantification of the signal intensity by the FLA-5000 imaging system and MultiGauge software (FujiFilm) is shown. The signal intensity in arbitrary units was plotted against the distance of migration in the gel. ( C ) rDNA copy number is slightly increased in the HMO1 deletion strain. rDNA copy number was determined in genomic DNA isolated from yeast strains carrying either a wild-type ( HMO1 , y881) or a complete deletion (Δ, y1159) of the HMO1 gene. The DNA was analyzed by quantitative Southern blot analysis of XcmI restriction fragments detecting either a 4.9-kb fragment of the promoter and coding region of the 35S rRNA gene (RDNp) or a 6.8-kb fragment containing the GAL gene locus ( GAL1 ). Signal intensities of the respective restriction fragments were determined using the FLA-5000 imaging system and MultiGauge software (FujiFilm). The same DNA was analyzed by qPCR amplifying either a region of the 18S rDNA or a region of the single-gene PHO5 locus. The primary data of the Southern blot analysis is shown, with the name of the probes used for hybridization indicated on the left . The diagram is a graphical representation of the results derived by the two kinds of analyses. The relative rDNA copy number was determined by normalizing the ratio of the amount of rDNA to the ratio of the amount of the single-copy genes. ( D ) Deletion of Hmo1 does not affect transcription factor binding at rDNA. ChEC was performed with nuclei isolated from yeast strains carrying either a wild-type ( HMO1 ) or a complete deletion (Δ) of the HMO1 gene and expressing the MNase fusion protein indicated at the top of the panel. Incubation in calcium-containing buffer was performed for the minutes depicted above each lane. DNA was analyzed as described in the legend for A cartoon of the genomic region is depicted on the right as described in the legend for An arrow points to the uncut XcmI fragment of the genomic DNA region.
Figure Legend Snippet: Hmo1 is a structural constituent of actively transcribed 35S rDNA chromatin. ( A ) Hmo1 remains associated with rDNA in the absence of Pol I. ChEC was performed with nuclei isolated from yeast strains cultured for 2 h at 37°C, carrying either a wild-type ( RRN3 ) or a temperature-sensitive ( rrn3 -ts) allele of the essential, Pol I-specific initiation factor Rrn3, and expressing the MNase fusion protein indicated at the top of the panel. Isolated nuclei were incubated in calcium-containing buffer at 30°C for the minutes depicted above each lane. DNA was isolated, digested with XcmI, and analyzed in a Southern blot as described in the legend for A cartoon of the genomic region is depicted on the right as described in the legend for An arrow points to the uncut XcmI fragment of the genomic DNA region. Two fragments of unknown origin are labeled by asterisks. ( B ) S-band chromatin can be established in the absence of Hmo1. Psoralen cross-link analyses were performed with nuclei isolated from yeast strains carrying either a wild-type ( HMO1 , y881) or a complete deletion (Δ, y1159) of the HMO1 gene. DNA was isolated, digested with EcoRI, and analyzed in a Southern blot as described in the legend for A profile analysis after quantification of the signal intensity by the FLA-5000 imaging system and MultiGauge software (FujiFilm) is shown. The signal intensity in arbitrary units was plotted against the distance of migration in the gel. ( C ) rDNA copy number is slightly increased in the HMO1 deletion strain. rDNA copy number was determined in genomic DNA isolated from yeast strains carrying either a wild-type ( HMO1 , y881) or a complete deletion (Δ, y1159) of the HMO1 gene. The DNA was analyzed by quantitative Southern blot analysis of XcmI restriction fragments detecting either a 4.9-kb fragment of the promoter and coding region of the 35S rRNA gene (RDNp) or a 6.8-kb fragment containing the GAL gene locus ( GAL1 ). Signal intensities of the respective restriction fragments were determined using the FLA-5000 imaging system and MultiGauge software (FujiFilm). The same DNA was analyzed by qPCR amplifying either a region of the 18S rDNA or a region of the single-gene PHO5 locus. The primary data of the Southern blot analysis is shown, with the name of the probes used for hybridization indicated on the left . The diagram is a graphical representation of the results derived by the two kinds of analyses. The relative rDNA copy number was determined by normalizing the ratio of the amount of rDNA to the ratio of the amount of the single-copy genes. ( D ) Deletion of Hmo1 does not affect transcription factor binding at rDNA. ChEC was performed with nuclei isolated from yeast strains carrying either a wild-type ( HMO1 ) or a complete deletion (Δ) of the HMO1 gene and expressing the MNase fusion protein indicated at the top of the panel. Incubation in calcium-containing buffer was performed for the minutes depicted above each lane. DNA was analyzed as described in the legend for A cartoon of the genomic region is depicted on the right as described in the legend for An arrow points to the uncut XcmI fragment of the genomic DNA region.

Techniques Used: Isolation, Cell Culture, Expressing, Incubation, Southern Blot, Labeling, Imaging, Software, Migration, Real-time Polymerase Chain Reaction, Hybridization, Derivative Assay, Binding Assay

5) Product Images from "Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract"

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract

Journal:

doi: 10.1093/aob/mcr232

Profile of DNA isolated from the soluble chromatin: DNA was extracted from fractionated nucleosomes obtained from chromatin derived from MNase-treated wheat nuclei and analysed on 1 % agarose gel. The lane numbers (25–39) indicate fraction numbers.
Figure Legend Snippet: Profile of DNA isolated from the soluble chromatin: DNA was extracted from fractionated nucleosomes obtained from chromatin derived from MNase-treated wheat nuclei and analysed on 1 % agarose gel. The lane numbers (25–39) indicate fraction numbers.

Techniques Used: Isolation, Derivative Assay, Agarose Gel Electrophoresis

6) Product Images from "Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y"

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201002043

Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.
Figure Legend Snippet: Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.

Techniques Used: In Silico, Generated, Transfection, Plasmid Preparation, Hemagglutination Assay, Purification, Silver Staining, SDS Page, Binding Assay, Imaging, Confocal Microscopy, Construct, Stable Transfection

Related Articles

Centrifugation:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: All centrifugation steps before MNase treatment were performed at 3,200 g . .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM).

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: All fractionation steps were performed at 4 ° C. Cell pellets were suspended in a non-denaturing isosmotic buffer [10 mM PIPES pH 7.0, 3 mM MgCl2 , 100 mM NaCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 0.1 mM PMSF, 1mM EGTA, 1× Protease inhibitor cocktail EDTA-free (Roche), 15 μM MG132, 10 mM N-Ethylmaleimide and 20 μM PR-619 (LifeSensors)] and extracted in the same buffer with 0.5 % (v/v) Triton-x 100 for 5 min. .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Addition of (NH4 )2 SO4 to a final concentration of 250 mM was used to facilitate extraction of stably DNA-bound proteins.

Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
Article Snippet: Nuclei were resuspended (10 × 106 nuclei/ml) in NBR supplemented with RNaseA (20 µ g/ml) and incubated at 20 ºC for 5 min. Chromatin was fragmented for 30 min at 20 ºC using 0.133 U/μl microccocal nuclease (MNase - Boehringer units; SigmaAldrich - N3755-500UN; titrated to give predominantly mono-nucleosomes). .. Nuclei were resuspended (10 × 106 nuclei/ml) in NBR supplemented with RNaseA (20 µ g/ml) and incubated at 20 ºC for 5 min. Chromatin was fragmented for 30 min at 20 ºC using 0.133 U/μl microccocal nuclease (MNase - Boehringer units; SigmaAldrich - N3755-500UN; titrated to give predominantly mono-nucleosomes).

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Where indicated, cells were treated with HU at 2 mM for 9 h. Cells were re-suspended in 1 ml of osmotic buffer (10 mM Hepes-NaOH, pH 7.9, 0.2 M potassium acetate, 0.34 M sucrose, 10% glycerol, 1 mM dithiotreitol, 0.1% Triton X-100 and protease inhibitors) and the sample was incubated for 5 min on ice. .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C. .. Reaction was stopped by adding EGTA (2 mM final concentration) to the sample.

Article Title: Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer
Article Snippet: The cells were washed three times with PBS and then harvested in ChIP lysis buffer (50 mM Tris–HCl, pH7.6, 1 mM CaCl2 , 0.2% Triton X-100). .. DNA was digested to 150–300 bp by MNase (SIGMA) before extensive centrifugation. .. Four volume of ChIP dilution buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) was added to the supernatant.

Article Title: Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway
Article Snippet: Nuclei were collected by centrifugation (13,000 g ) and washed twice with 1 ml of buffer A [15 mM tris (pH 7.5), 15 mM NaCl, 60 mM KCl, 0.34 M sucrose, 0.5 mM spermidine, 0.15 mM spermine, 0.25 mM phenylmethylsulfonyl fluoride, and 0.1% β-mercaptoethanol]. .. CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C.

Article Title: Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes
Article Snippet: After being shaken for 3–5 h at 4°C in MNase lysis buffer, nuclei were collected by centrifugation and washed in ice-cold MNase digestion buffer without CaCl2 (10 mM Tris at pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, and 0.5 mM spermidine). .. Nuclei were treated with 1 unit of MNase (nuclease micrococcal from Staphylococcus aureus; N5386-200UN; Sigma-Aldrich) for 60–90 min at 25°C.

Blocking Assay:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Immunoprecipitations were carried out on nuclear extracts prepared from HEK 293T cells synchronized in S-phase with a single block in thymidine. .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Electrophoresis:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Samples were subjected to electrophoresis through 8% polyacrylamide-bis (29:1) gel. .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Incubation:

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract
Article Snippet: The strategy for preparation of donor chromatin was adapted from that described by with some modifications optimized for plant chromatin. .. Approximately 0·03 U MNase (Sigma-Aldrich, India) per milligram of DNA was added to the resuspended nuclei and incubated on ice for 2 min with intermittent mixing at an interval of 30 s. Nuclei were centrifuged at 8500 g for 5 min and the pellet was resuspended in nuclei digestion buffer containing 2 m m CaCl2 and the suspension was incubated at 37 °C in a water bath with continuous stirring for a further 2 min. EDTA was added at 0·5 m to stop MNase activity. .. The pellet obtained after centrifugation at 8500 g at 4 °C was resuspended in 15 m m Tris/HCl, pH 7·5, with 0·2 m m EDTA and incubated for 30 min on ice.

Article Title: Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules
Article Snippet: Nuclei isolated from formaldehyde-cross-linked yeast cells (strain NOY505) were digested as described previously ( ). .. For the experiment presented in , nuclei from ∼85 mg of cells (wet weight) were incubated in 200 μL of reaction buffer containing 5 U/mL MNase (Sigma) for 20 min at 37°C. .. ChEC was carried out using nuclei from formaldehyde-cross-linked yeast cells as described ( ) with slight modifications.

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: All fractionation steps were performed at 4 ° C. Cell pellets were suspended in a non-denaturing isosmotic buffer [10 mM PIPES pH 7.0, 3 mM MgCl2 , 100 mM NaCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 0.1 mM PMSF, 1mM EGTA, 1× Protease inhibitor cocktail EDTA-free (Roche), 15 μM MG132, 10 mM N-Ethylmaleimide and 20 μM PR-619 (LifeSensors)] and extracted in the same buffer with 0.5 % (v/v) Triton-x 100 for 5 min. .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Addition of (NH4 )2 SO4 to a final concentration of 250 mM was used to facilitate extraction of stably DNA-bound proteins.

Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
Article Snippet: Nuclei were pelleted at 2,000 g for 3 min at 4 ºC, then washed with NBR buffer (85 mM NaCl, 5.5 % Sucrose, 10 mM TrisHCl pH 7.5, 3 mM MgCl2 , 1.5 mM CaCl2 , 0.2 mM PMSF and 1 mM DTT) and pelleted at 2,000 g for 3 min at 4 ºC. .. Nuclei were resuspended (10 × 106 nuclei/ml) in NBR supplemented with RNaseA (20 µ g/ml) and incubated at 20 ºC for 5 min. Chromatin was fragmented for 30 min at 20 ºC using 0.133 U/μl microccocal nuclease (MNase - Boehringer units; SigmaAldrich - N3755-500UN; titrated to give predominantly mono-nucleosomes). .. Digestion was stopped with the addition of an equal volume of STOP bufffer (215 mM NaCl, 10 mM TrisHCl pH 8, 20 mM EDTA, 5.5 %, Sucrose, 2 % TritonX 100, 0.2 mM PMSF, 1 mM DTT, 2X Protease Inhibitors) and digested nuclei left on ice overnight to release soluble, fragmented chromatin.

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Where indicated, cells were treated with HU at 2 mM for 9 h. Cells were re-suspended in 1 ml of osmotic buffer (10 mM Hepes-NaOH, pH 7.9, 0.2 M potassium acetate, 0.34 M sucrose, 10% glycerol, 1 mM dithiotreitol, 0.1% Triton X-100 and protease inhibitors) and the sample was incubated for 5 min on ice. .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Article Title: Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer
Article Snippet: DNA was digested to 150–300 bp by MNase (SIGMA) before extensive centrifugation. .. DNA was digested to 150–300 bp by MNase (SIGMA) before extensive centrifugation.

Article Title: Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin
Article Snippet: Cells were grown to 70–80% confluence and dishes were washed once with 5 ml PBS. .. Cells were incubated for 90 s with 3 ml of permeabilisation buffer (15 mM Tris–HCl pH 7.6, 300 mM saccharose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% (v/v) NP40 and fresh 0.5 mM 2-mercaptoethanol) including 100–2000 units of MNase (Sigma) ( ). .. The DNA hydrolysis reaction was stopped by the addition of 3 ml of stop buffer (50 mM Tris–HCl pH 8, 20 mM EDTA and 1% SDS).

Article Title: Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway
Article Snippet: Nuclei were resuspended in buffer A and aliquoted into 100 μl of aliquots. .. CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C. .. Each aliquot was put back on ice at different time points, and digestion was immediately stopped by addition of 3 μl of EDTA.

Article Title: Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations
Article Snippet: Spermatozoa (100 million) were resuspended in 1 ml of 100 mM dithiothreitol (DTT; Sigma-Aldrich) in PBS and incubated for 2 h on a wheel at room temperature. .. After 30 min, 200 μl of MNase buffer (sucrose was added at a 0.3 M final concentration to the MNase buffer stock (85 mM Tris-HCl, pH 7.5, 3 mM MgCl2 and 2 mM CaCl2 ) and 60 units of MNase (Sigma-Aldrich) for every four million sperm to each of the tubes (200 μl/4 M cells) and vortexed.

Article Title: BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis
Article Snippet: The pellet was then washed in 1ml lysis buffer with calcium (1 mM CaCl2), centrifuged at 150 g for 10 minutes at 4°C and re-suspended in 50μl lysis buffer with calcium. .. To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C. .. The digestion was stopped by adding 18 μl of 0.5 M EDTA and 14 μl 0.1M EGTA.

Article Title: Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)
Article Snippet: For nuclease-mediated solubilisation of chromatin-bound proteins 2 × 106 ESCs were used for each reaction. .. MNase digest: chromatin was incubated in MNase buffer with 1 U MNase (Sigma N3755)/100 μl reaction volume at 28 °C for 1,1.5 and 2 h. The reaction was stopped with 5 μl 200 mM EGTA. .. DNAse I digest: chromatin was incubated in DNAse buffer with 50 U DNAse-I (Roche, 04536282001)/100 μl reaction volume for 2 h at 37 °C.

Activity Assay:

Article Title: Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract
Article Snippet: The strategy for preparation of donor chromatin was adapted from that described by with some modifications optimized for plant chromatin. .. Approximately 0·03 U MNase (Sigma-Aldrich, India) per milligram of DNA was added to the resuspended nuclei and incubated on ice for 2 min with intermittent mixing at an interval of 30 s. Nuclei were centrifuged at 8500 g for 5 min and the pellet was resuspended in nuclei digestion buffer containing 2 m m CaCl2 and the suspension was incubated at 37 °C in a water bath with continuous stirring for a further 2 min. EDTA was added at 0·5 m to stop MNase activity. .. The pellet obtained after centrifugation at 8500 g at 4 °C was resuspended in 15 m m Tris/HCl, pH 7·5, with 0·2 m m EDTA and incubated for 30 min on ice.

Cell Fractionation:

Article Title: Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)
Article Snippet: Cell fractionation and solubilisation of chromatin-bound proteins by salt extraction was performed as described in ref. . .. MNase digest: chromatin was incubated in MNase buffer with 1 U MNase (Sigma N3755)/100 μl reaction volume at 28 °C for 1,1.5 and 2 h. The reaction was stopped with 5 μl 200 mM EGTA.

Modification:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min).

Western Blot:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: The gel was electro-blotted onto a PVDF membrane and proteins on the blot were detected with a monoclonal horseradish peroxidase-conjugated anti-Flag antibody (Abcam, ab49763) detected using the ECL+ system (GE Healthcare). .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Derivative Assay:

Article Title: Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription
Article Snippet: Chromatin for MNase digestion was prepared essentially as described ( ). .. Spheroplasts derived from 100-mL cultures were treated with either 0.5 U (underdigested) or 2 U (overdigested) of MNase (Sigma) for 45 min at 37°C. .. Purified and precipitated DNA was sequenced using the paired-end TruSeq protocol (Illumina).

Immunoprecipitation:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: Paragraph title: Immunoprecipitation experiments ... After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Protease Inhibitor:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: All centrifugation steps before MNase treatment were performed at 3,200 g . .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: All fractionation steps were performed at 4 ° C. Cell pellets were suspended in a non-denaturing isosmotic buffer [10 mM PIPES pH 7.0, 3 mM MgCl2 , 100 mM NaCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 0.1 mM PMSF, 1mM EGTA, 1× Protease inhibitor cocktail EDTA-free (Roche), 15 μM MG132, 10 mM N-Ethylmaleimide and 20 μM PR-619 (LifeSensors)] and extracted in the same buffer with 0.5 % (v/v) Triton-x 100 for 5 min. .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min). .. Addition of (NH4 )2 SO4 to a final concentration of 250 mM was used to facilitate extraction of stably DNA-bound proteins.

Article Title: Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations
Article Snippet: After 30 min, 200 μl of MNase buffer (sucrose was added at a 0.3 M final concentration to the MNase buffer stock (85 mM Tris-HCl, pH 7.5, 3 mM MgCl2 and 2 mM CaCl2 ) and 60 units of MNase (Sigma-Aldrich) for every four million sperm to each of the tubes (200 μl/4 M cells) and vortexed. .. Chromatin (1 ml) was dispensed into 1.5 ml tubes and 5 μg of each ChIP grade antibody added: H3K4me3 (Abcam, Cambridge, UK), H3K4me1, H3K27me3 (Millipore, Hertfordshire, UK), H3K9me3 (Abcam), H3 (Abcam) or Ig rabbit control (Abcam).

Cell Culture:

Article Title: Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin
Article Snippet: Cells were incubated at 37°C with 5% CO2 on 15 cm cell culture dishes. .. Cells were incubated for 90 s with 3 ml of permeabilisation buffer (15 mM Tris–HCl pH 7.6, 300 mM saccharose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% (v/v) NP40 and fresh 0.5 mM 2-mercaptoethanol) including 100–2000 units of MNase (Sigma) ( ).

Generated:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: All centrifugation steps before MNase treatment were performed at 3,200 g . .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

other:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: MNase and all chemicals were purchased from Sigma-Aldrich unless otherwise specified.

Imaging:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Magnetic beads (Invitrogen) were washed four times with buffer C (20 mM Hepes/KOH, pH 7.9, 20% [vol/vol] glycerol, 0.2 mM EDTA, 0.2% [vol/vol] Triton X-100, 300 mM KCl, and protease inhibitor cocktail [Roche]).

Sequencing:

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin
Article Snippet: For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin. .. DNA was purified and analysed by a primer extension reaction (denaturation, 5 min at 95°C; annealing, 2 min at 56°C; extension, 1 min at 72°C) using radioactively labelled oligonucleotides that hybridized to the rDNA promoter.

Sonication:

Article Title: The Heterochromatin Landscape in Migrating Cells and the Importance of H3K27me3 for Associated Transcriptome Alterations
Article Snippet: Following cross linking with 1% PFA, cells were collected and aliquoted to 107 cells. .. Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer. .. IP was done using magnetic protein A/G beads (BioVision, 6527-1, Milpitas, CA, USA) coupled to 24 µL of rabbit anti-H3K9me3 (Millipore, 07-442, Temecula, CA, USA), 12 µL of rabbit anti-H3K27me3 (Millipore, 07-449, Temecula, CA, USA), or 24 µL of rabbit anti-H4K20me1 (Millipore, 17-610, Temecula, CA, USA).

ChIP-sequencing:

Article Title: Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression
Article Snippet: Paragraph title: Nucleosome-resolution ChIP-seq assays ... The MCF7 breast cancer cells were treated with MNase (Sigma N3755) and ChIP was done with H3K4me2 antibody (Millipore CMA303) as previously published .

Article Title: The Heterochromatin Landscape in Migrating Cells and the Importance of H3K27me3 for Associated Transcriptome Alterations
Article Snippet: Paragraph title: 2.2. ChIP-seq ... Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer.

DNA Extraction:

Article Title: Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin
Article Snippet: Paragraph title: Differential MNase hydrolysis and DNA isolation ... Cells were incubated for 90 s with 3 ml of permeabilisation buffer (15 mM Tris–HCl pH 7.6, 300 mM saccharose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% (v/v) NP40 and fresh 0.5 mM 2-mercaptoethanol) including 100–2000 units of MNase (Sigma) ( ).

Sensitive Assay:

Article Title: Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway
Article Snippet: Paragraph title: MNase digestion sensitivity assay ... CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C.

Magnetic Beads:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Article Title: Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations
Article Snippet: After 30 min, 200 μl of MNase buffer (sucrose was added at a 0.3 M final concentration to the MNase buffer stock (85 mM Tris-HCl, pH 7.5, 3 mM MgCl2 and 2 mM CaCl2 ) and 60 units of MNase (Sigma-Aldrich) for every four million sperm to each of the tubes (200 μl/4 M cells) and vortexed. .. The reaction was stopped by adding 4 μl of EDTA 0.5 M, vortexing and placing on ice for at least 5 min followed by centrifugation for 10 min at maximum speed at room temperature.

Isolation:

Article Title: Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules
Article Snippet: Nuclei isolated from formaldehyde-cross-linked yeast cells (strain NOY505) were digested as described previously ( ). .. For the experiment presented in , nuclei from ∼85 mg of cells (wet weight) were incubated in 200 μL of reaction buffer containing 5 U/mL MNase (Sigma) for 20 min at 37°C.

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM).

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Crude chromatin was isolated after Triton-X 100 extraction and MNase digestion. .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min).

Article Title: The Heterochromatin Landscape in Migrating Cells and the Importance of H3K27me3 for Associated Transcriptome Alterations
Article Snippet: Following cross linking with 1% PFA, cells were collected and aliquoted to 107 cells. .. Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer. .. IP was done using magnetic protein A/G beads (BioVision, 6527-1, Milpitas, CA, USA) coupled to 24 µL of rabbit anti-H3K9me3 (Millipore, 07-442, Temecula, CA, USA), 12 µL of rabbit anti-H3K27me3 (Millipore, 07-449, Temecula, CA, USA), or 24 µL of rabbit anti-H4K20me1 (Millipore, 17-610, Temecula, CA, USA).

Article Title: BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis
Article Snippet: Paragraph title: Chromatin isolation from pachytene spermatocytes and MNase digestion of native chromatin to obtain a mononucleosome fraction ... To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C.

Article Title: Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)
Article Snippet: Paragraph title: ESCs chromatin isolation and Rif1 solubilisation test ... MNase digest: chromatin was incubated in MNase buffer with 1 U MNase (Sigma N3755)/100 μl reaction volume at 28 °C for 1,1.5 and 2 h. The reaction was stopped with 5 μl 200 mM EGTA.

Purification:

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin
Article Snippet: Chromatin assembly and purification, nucleosome mapping and nucleosome remodelling reactions were performed as described in the Supplementary Data . .. For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin. .. Reactions were stopped by the addition of 0.2 volumes of stop buffer [4% sodium dodecyl sulfate (SDS), 100 mM EDTA].

Article Title: Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin
Article Snippet: Cells were incubated for 90 s with 3 ml of permeabilisation buffer (15 mM Tris–HCl pH 7.6, 300 mM saccharose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% (v/v) NP40 and fresh 0.5 mM 2-mercaptoethanol) including 100–2000 units of MNase (Sigma) ( ). .. RNA was digested by the addition of 250 μg RNase A and incubation for 2 h. Subsequently 250 μg of proteinase K were added to the culture dishes and incubated over night at 37°C.

Article Title: The Heterochromatin Landscape in Migrating Cells and the Importance of H3K27me3 for Associated Transcriptome Alterations
Article Snippet: Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer. .. Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer.

Article Title: Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway
Article Snippet: CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C. .. CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C.

Article Title: BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis
Article Snippet: To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C. .. To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C.

Article Title: Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes
Article Snippet: Nuclei were treated with 1 unit of MNase (nuclease micrococcal from Staphylococcus aureus; N5386-200UN; Sigma-Aldrich) for 60–90 min at 25°C. .. Reactions were stopped by the addition of 80 μl MNase digestion buffer with CaCl2 , 20 μl MNase stop buffer (100 mM EDTA and 10 mM EGTA), 75 μg proteinase K, and 20 μl 10% SDS, and then cells were incubated at 65°C overnight.

Polymerase Chain Reaction:

Article Title: Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway
Article Snippet: CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C. .. CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C.

Article Title: BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis
Article Snippet: To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C. .. To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C.

Staining:

Article Title: Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin
Article Snippet: Cells were incubated for 90 s with 3 ml of permeabilisation buffer (15 mM Tris–HCl pH 7.6, 300 mM saccharose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% (v/v) NP40 and fresh 0.5 mM 2-mercaptoethanol) including 100–2000 units of MNase (Sigma) ( ). .. Fragmented DNA was purified by ammonium acetate and ethanol precipitation.

Silver Staining:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Magnetic beads (Invitrogen) were washed four times with buffer C (20 mM Hepes/KOH, pH 7.9, 20% [vol/vol] glycerol, 0.2 mM EDTA, 0.2% [vol/vol] Triton X-100, 300 mM KCl, and protease inhibitor cocktail [Roche]).

Chromatin Immunoprecipitation:

Article Title: An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites
Article Snippet: Procedure: Harvest cells; spin down at 600 g 3 min in swing bucket rotor (for mammalian cells, 5–10 × 106 cells are typically used per ChIP in a 1 ml MNase reaction). .. Place on ice for 10 min Scale up if more than 10 million cells Pre-warm tubes by placing in 37°C water bath for 2 min. Add pre-determined amount of MNase (Sigma N3755 @ 0.2 units per µl); incubate at 37°C Stop by adding EDTA to 10 mM and EGTA to 20 mM; make a master mix to allow faster addition.

Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
Article Snippet: Paragraph title: Native Chromatin Immunoprecipitation ... Nuclei were resuspended (10 × 106 nuclei/ml) in NBR supplemented with RNaseA (20 µ g/ml) and incubated at 20 ºC for 5 min. Chromatin was fragmented for 30 min at 20 ºC using 0.133 U/μl microccocal nuclease (MNase - Boehringer units; SigmaAldrich - N3755-500UN; titrated to give predominantly mono-nucleosomes).

Article Title: Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer
Article Snippet: Paragraph title: ChIP assay ... DNA was digested to 150–300 bp by MNase (SIGMA) before extensive centrifugation.

Article Title: Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression
Article Snippet: The modulator quotient null distribution over the set of MRVSs is then used as in VSE to calculate a p-value for the enrichment of the AVS in affinity modulating SNPs. .. The MCF7 breast cancer cells were treated with MNase (Sigma N3755) and ChIP was done with H3K4me2 antibody (Millipore CMA303) as previously published . .. Libraries were prepared for Illumina Genome Analyzer according to manufactures instructions.

Article Title: The Heterochromatin Landscape in Migrating Cells and the Importance of H3K27me3 for Associated Transcriptome Alterations
Article Snippet: Chromatin immunoprecipitation (ChIP) was performed as described previously [ ] with the following modifications. .. Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer.

Article Title: Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations
Article Snippet: Paragraph title: ChIP protocol ... After 30 min, 200 μl of MNase buffer (sucrose was added at a 0.3 M final concentration to the MNase buffer stock (85 mM Tris-HCl, pH 7.5, 3 mM MgCl2 and 2 mM CaCl2 ) and 60 units of MNase (Sigma-Aldrich) for every four million sperm to each of the tubes (200 μl/4 M cells) and vortexed.

SDS Page:

Article Title: Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
Article Snippet: The beads were then washed three times with 600 μl of washing buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 300 mM NaCl, 1 mM dithiotreitol, 0.1% Nonidet-P40) and re-suspended in 30 μl of SDS-PAGE loading buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 200 mM β-mercaptoethanol, 0.5% SDS, 0.01% blue bromophenol). .. After centrifugation (800 × g for 5 min), the nucleus/chromatin fraction in the pellet was re-suspended in 1 ml of hypotonic buffer (10 mM Hepes-NaOH, pH 7.9, 0.25 M potassium acetate, 1 mM dithiothreitol, 0.1% Triton X-100 and protease inhibitors) containing 10 mM CaCl2 and microccocal nuclease (0.2 units/μl; SIGMA, cat. N3755) for 20 min at 37°C.

Software:

Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin
Article Snippet: For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin. .. DNA was purified and analysed by a primer extension reaction (denaturation, 5 min at 95°C; annealing, 2 min at 56°C; extension, 1 min at 72°C) using radioactively labelled oligonucleotides that hybridized to the rDNA promoter.

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Magnetic beads (Invitrogen) were washed four times with buffer C (20 mM Hepes/KOH, pH 7.9, 20% [vol/vol] glycerol, 0.2 mM EDTA, 0.2% [vol/vol] Triton X-100, 300 mM KCl, and protease inhibitor cocktail [Roche]).

Article Title: Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression
Article Snippet: The MCF7 breast cancer cells were treated with MNase (Sigma N3755) and ChIP was done with H3K4me2 antibody (Millipore CMA303) as previously published . .. The MCF7 breast cancer cells were treated with MNase (Sigma N3755) and ChIP was done with H3K4me2 antibody (Millipore CMA303) as previously published .

Real-time Polymerase Chain Reaction:

Article Title: BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis
Article Snippet: To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C. .. Finally, DNA from the mononucleosome fractions was extracted and purified using a QIAquick PCR purification kit (Qiagen).

Article Title: Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes
Article Snippet: Nuclei were treated with 1 unit of MNase (nuclease micrococcal from Staphylococcus aureus; N5386-200UN; Sigma-Aldrich) for 60–90 min at 25°C. .. Nuclei were treated with 1 unit of MNase (nuclease micrococcal from Staphylococcus aureus; N5386-200UN; Sigma-Aldrich) for 60–90 min at 25°C.

Agarose Gel Electrophoresis:

Article Title: Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway
Article Snippet: CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C. .. CaCl2 (1.2 μl of 0.1 M) was added to each aliquot, and nuclei were digested by addition of 0.25 μl of MNase (200 U/ml; Sigma-Aldrich) and incubated at 30°C.

Article Title: Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes
Article Snippet: Nuclei were treated with 1 unit of MNase (nuclease micrococcal from Staphylococcus aureus; N5386-200UN; Sigma-Aldrich) for 60–90 min at 25°C. .. Reactions were stopped by the addition of 80 μl MNase digestion buffer with CaCl2 , 20 μl MNase stop buffer (100 mM EDTA and 10 mM EGTA), 75 μg proteinase K, and 20 μl 10% SDS, and then cells were incubated at 65°C overnight.

Ethanol Precipitation:

Article Title: Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin
Article Snippet: Cells were incubated for 90 s with 3 ml of permeabilisation buffer (15 mM Tris–HCl pH 7.6, 300 mM saccharose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% (v/v) NP40 and fresh 0.5 mM 2-mercaptoethanol) including 100–2000 units of MNase (Sigma) ( ). .. RNA was digested by the addition of 250 μg RNase A and incubation for 2 h. Subsequently 250 μg of proteinase K were added to the culture dishes and incubated over night at 37°C.

Concentration Assay:

Article Title: An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites
Article Snippet: Place on ice for 10 min Scale up if more than 10 million cells Pre-warm tubes by placing in 37°C water bath for 2 min. Add pre-determined amount of MNase (Sigma N3755 @ 0.2 units per µl); incubate at 37°C Stop by adding EDTA to 10 mM and EGTA to 20 mM; make a master mix to allow faster addition. .. Place on ice for 10 min Scale up if more than 10 million cells Pre-warm tubes by placing in 37°C water bath for 2 min. Add pre-determined amount of MNase (Sigma N3755 @ 0.2 units per µl); incubate at 37°C Stop by adding EDTA to 10 mM and EGTA to 20 mM; make a master mix to allow faster addition.

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: All centrifugation steps before MNase treatment were performed at 3,200 g . .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM). .. Centrifugation was performed at 20,000 g for 20 min. Supernatants of four MNase digests were combined, and salt concentration was adjusted to 150 mM KCl.

Article Title: Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations
Article Snippet: The cells were aliquotted in 100-μL aliquots with 100 μl of complete buffer 1 with detergent (15 mM Tris-HCl (pH 7.5), 60 mM KCl, 5 mM MgCl2 , 0.1 mM EGTA, 0.3 M sucrose, 10 mM DTT, 0.5% (vol/vol) NP-40 and 1% (wt/vol) deoxycholate). .. After 30 min, 200 μl of MNase buffer (sucrose was added at a 0.3 M final concentration to the MNase buffer stock (85 mM Tris-HCl, pH 7.5, 3 mM MgCl2 and 2 mM CaCl2 ) and 60 units of MNase (Sigma-Aldrich) for every four million sperm to each of the tubes (200 μl/4 M cells) and vortexed. .. The reaction was stopped by adding 4 μl of EDTA 0.5 M, vortexing and placing on ice for at least 5 min followed by centrifugation for 10 min at maximum speed at room temperature.

Article Title: BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis
Article Snippet: To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C. .. Finally, DNA from the mononucleosome fractions was extracted and purified using a QIAquick PCR purification kit (Qiagen).

Article Title: Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes
Article Snippet: Cells were cross linked with 1% formaldehyde for 3 min at 37°C, and the reaction was quenched by the addition of glycine to a final concentration of 0.125 M. Cells were then washed once with PBS and scraped in ice-cold MNase NP-40 lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2 , 0.5% NP-40, 0.15 mM spermine, and 0.5 mM spermidine). .. Nuclei were treated with 1 unit of MNase (nuclease micrococcal from Staphylococcus aureus; N5386-200UN; Sigma-Aldrich) for 60–90 min at 25°C.

Fractionation:

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Paragraph title: Preparation of whole cell lysates, Chromatin fractionation, and Immunoprecipitations ... Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min).

Magnetic Cell Separation:

Article Title: Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression
Article Snippet: The MCF7 breast cancer cells were treated with MNase (Sigma N3755) and ChIP was done with H3K4me2 antibody (Millipore CMA303) as previously published . .. The MCF7 breast cancer cells were treated with MNase (Sigma N3755) and ChIP was done with H3K4me2 antibody (Millipore CMA303) as previously published .

DNA Purification:

Article Title: Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer
Article Snippet: DNA was digested to 150–300 bp by MNase (SIGMA) before extensive centrifugation. .. The beads were washed five times and DNA was eluted by Chip elution buffer (0.1 M NaHCO3 , 1% SDS, 20 μg/ml proteinase K).

Lysis:

Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
Article Snippet: Mononucleosomes for IP experiments were generated as described previously , with the following changes: For cell lysis, 0.1% NP-40 was used instead of Triton X-100. .. Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM).

Article Title: The core spliceosome as target and effector of non-canonical ATM signaling
Article Snippet: Whole cell lysates were prepared by lysis of equal cell numbers in 60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue. .. Following centrifugation (650g, 5 min), nuclei depleted from soluble nucleoplasm were washed with MNase digestion buffer [50 mM Tris-Cl pH 7.5, 4 mM MgCl2 , 50 mM KCl, 300 mM Sucrose, 0.5 mM Na2 VO4 , 5mM NaF, 5mM Na4 P2 O7 , 10mM beta-glycerolphosphate, 1mM PMSF, 1mM, EGTA and 1× EDTA-free Protease inhibitor cocktail] and subsequently incubated with 0.3 U MNase (Sigma)/1×106 nuclei, and 1 mM CaCl2 (37° C, 10 min).

Article Title: Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer
Article Snippet: The cells were washed three times with PBS and then harvested in ChIP lysis buffer (50 mM Tris–HCl, pH7.6, 1 mM CaCl2 , 0.2% Triton X-100). .. DNA was digested to 150–300 bp by MNase (SIGMA) before extensive centrifugation.

Article Title: The Heterochromatin Landscape in Migrating Cells and the Importance of H3K27me3 for Associated Transcriptome Alterations
Article Snippet: Following cross linking with 1% PFA, cells were collected and aliquoted to 107 cells. .. Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer. .. IP was done using magnetic protein A/G beads (BioVision, 6527-1, Milpitas, CA, USA) coupled to 24 µL of rabbit anti-H3K9me3 (Millipore, 07-442, Temecula, CA, USA), 12 µL of rabbit anti-H3K27me3 (Millipore, 07-449, Temecula, CA, USA), or 24 µL of rabbit anti-H4K20me1 (Millipore, 17-610, Temecula, CA, USA).

Article Title: BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis
Article Snippet: The pellet was then washed in 1ml lysis buffer with calcium (1 mM CaCl2), centrifuged at 150 g for 10 minutes at 4°C and re-suspended in 50μl lysis buffer with calcium. .. To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase (N5386-50U, Sigma-Aldrich) (0.125 U/μl, 0.25 U/μl, 0.5 U/μl, 1 U/μl, 2 U/μl, 3 U/μl), followed by 10 minutes incubation at 37°C.

Article Title: Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes
Article Snippet: After being shaken for 3–5 h at 4°C in MNase lysis buffer, nuclei were collected by centrifugation and washed in ice-cold MNase digestion buffer without CaCl2 (10 mM Tris at pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, and 0.5 mM spermidine). .. Nuclei were treated with 1 unit of MNase (nuclease micrococcal from Staphylococcus aureus; N5386-200UN; Sigma-Aldrich) for 60–90 min at 25°C.

Gel Extraction:

Article Title: The Heterochromatin Landscape in Migrating Cells and the Importance of H3K27me3 for Associated Transcriptome Alterations
Article Snippet: Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer. .. Nuclei were isolated by lysis buffer and DNA was collected following fragmentation by 0.7 mU/μL of MNase (N3755, Sigma-Aldrich, Rehovot, Israel) at 37 °C for 15 min that was followed by a brief sonication in RIPA buffer.

Article Title: Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes
Article Snippet: Nuclei were treated with 1 unit of MNase (nuclease micrococcal from Staphylococcus aureus; N5386-200UN; Sigma-Aldrich) for 60–90 min at 25°C. .. Reactions were stopped by the addition of 80 μl MNase digestion buffer with CaCl2 , 20 μl MNase stop buffer (100 mM EDTA and 10 mM EGTA), 75 μg proteinase K, and 20 μl 10% SDS, and then cells were incubated at 65°C overnight.

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    Millipore mnase1
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