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  • 95
    Millipore microccocal nuclease digestion
    Microccocal Nuclease Digestion, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche micrococcal nuclease s7 mnase
    Bulk chromatin and centromeric chromatin were solubilized by <t>MNase</t> digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at <t>37°C.</t> Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.
    Micrococcal Nuclease S7 Mnase, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher mnase
    Digestion of different regions of the rDNA units with <t>MNase.</t> (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at <t>24°C</t> for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was
    Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore mnase
    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 <t>mononucleosomes</t> 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 <t>MNase-treated</t> 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.
    Mnase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs mnase
    BEN-2 shows B cell-specific <t>nucleosome</t> occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with <t>MNase</t> digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.
    Mnase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1062 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    BioShop micrococcal nuclease s7
    BEN-2 shows B cell-specific <t>nucleosome</t> occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with <t>MNase</t> digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.
    Micrococcal Nuclease S7, supplied by BioShop, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Worthington Biochemical mnase
    Expression of mutant <t>H2B</t> in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of <t>MNase</t> sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.
    Mnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 93/100, based on 809 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche mnase buffer
    Expression of mutant <t>H2B</t> in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of <t>MNase</t> sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.
    Mnase Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    US Biological Life Sciences p1 microccocal nuclease
    Expression of mutant <t>H2B</t> in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of <t>MNase</t> sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.
    P1 Microccocal Nuclease, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc mnase
    Expression of mutant <t>H2B</t> in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of <t>MNase</t> sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.
    Mnase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa mnase
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim mnase
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    New England Biolabs 1x mnase buffer
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    1x Mnase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Active Motif mnase cocktail
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase Cocktail, supplied by Active Motif, used in various techniques. Bioz Stars score: 86/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore mnase i
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher mnase enzyme
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa mnase i
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche mnase s7
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase S7, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher mnase i
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher usb mnase
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Usb Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Diagenode mnase
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase, supplied by Diagenode, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc mnase
    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and <t>MNase</t> digestion. The genomic <t>DNA</t> was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.
    Mnase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

    Journal: Molecular and Cellular Biology

    Article Title: CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells

    doi: 10.1128/MCB.22.7.2229-2241.2002

    Figure Lengend Snippet: Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

    Article Snippet: The nuclear suspension was digested with MNase (Roche Diagnostics) at 37°C after addition of CaCl2 to a final concentration of 2 mM.

    Techniques: Isolation, Western Blot, Centrifugation, Incubation, Sonication, Polyacrylamide Gel Electrophoresis, Immunostaining, Marker, Agarose Gel Electrophoresis, Staining, Immunolabeling, Recombinant

    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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01409-06

    Figure Lengend 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

    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.

    Techniques: Isolation, Purification

    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.

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201002043

    Figure Lengend 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.

    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).

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

    BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.

    Journal: bioRxiv

    Article Title: Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary and enhancer elements in B cells

    doi: 10.1101/2020.02.16.941070

    Figure Lengend Snippet: BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.

    Article Snippet: To assess nucleosome occupancy, 1000 Gel Units MNase (New England Biolabs) was used.

    Techniques: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.

    Journal: Cancer discovery

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    doi: 10.1158/2159-8290.CD-19-0393

    Figure Lengend Snippet: Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.

    Article Snippet: MNase susceptibility assays were performed on nucleosomes made with WT, E76Q and E76K H2B mutants by mixing each nucleosome (0.4 pmol) with 9.6U of MNase (Worthington) and incubating for indicated amount of time.

    Techniques: Expressing, Mutagenesis, Incubation, Marker, Reverse Transcription Polymerase Chain Reaction, Purification, Real-time Polymerase Chain Reaction

    The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.

    Journal: Cancer discovery

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    doi: 10.1158/2159-8290.CD-19-0393

    Figure Lengend Snippet: The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.

    Article Snippet: MNase susceptibility assays were performed on nucleosomes made with WT, E76Q and E76K H2B mutants by mixing each nucleosome (0.4 pmol) with 9.6U of MNase (Worthington) and incubating for indicated amount of time.

    Techniques: Mutagenesis, In Vitro, Recombinant, Filtration, Chromatography, Clear Native PAGE, Migration, Sensitive Assay

    H2B-E76K fundamentally alters chromatin structure and dynamics. (A) Micrococcal nuclease (MNase) assay with nuclei of MCF10A cells stably expressing either WT or H2B-E76K demonstrate that E76K expression significantly increases sensitivity to MNase. Digest efficiency was visualized by agarose gel. (B) Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that H2B-E76K has significantly faster chromatin dynamics than WT. FRAP analysis was carried out after induction of either WT or E76K mutant H2B-GFP fusions for 5 or 35 days in MCF10A cells. Dashed lines represent cells expressing H2B-E76K, solid lines represent data from cells expressing inducible GFP-tagged WT H2B (n = 10 cells). (C) Representative FRAP assay pre-bleach, bleach and post-bleach images of nuclei expressing GFP-tagged WT or H2B-E76K indicate faster fluorescent recovery in cells expressing E76K. (D) FRAP analysis of histone H2A-GFP dynamics in MCF10A cells expressing only H2A-GFP (n = 20 cells) or both H2A-GFP and a mutant H2B E76K mCherry fusion (n = 25 cells) with standard deviation envelopes. Inset demonstrates co-expression of H2A GFP and E76K mCherry. (E) Representative pre-bleach, bleach and post-bleach images of H2A GFP in WT MCF10A cells or in cells co-expressing mCherry tagged H2B-E76K. (F) 100nm particles injected into the nucleus had significantly increased mean square displacement (MSD) over time in MCF10A cells stably expressing exogenous H2B-E76K (red) compared to cells expressing WT H2B (blue). N=68 (WT) and N=67 (E76K) cells were analyzed. Error bars represent SEM.

    Journal: Cancer discovery

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    doi: 10.1158/2159-8290.CD-19-0393

    Figure Lengend Snippet: H2B-E76K fundamentally alters chromatin structure and dynamics. (A) Micrococcal nuclease (MNase) assay with nuclei of MCF10A cells stably expressing either WT or H2B-E76K demonstrate that E76K expression significantly increases sensitivity to MNase. Digest efficiency was visualized by agarose gel. (B) Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that H2B-E76K has significantly faster chromatin dynamics than WT. FRAP analysis was carried out after induction of either WT or E76K mutant H2B-GFP fusions for 5 or 35 days in MCF10A cells. Dashed lines represent cells expressing H2B-E76K, solid lines represent data from cells expressing inducible GFP-tagged WT H2B (n = 10 cells). (C) Representative FRAP assay pre-bleach, bleach and post-bleach images of nuclei expressing GFP-tagged WT or H2B-E76K indicate faster fluorescent recovery in cells expressing E76K. (D) FRAP analysis of histone H2A-GFP dynamics in MCF10A cells expressing only H2A-GFP (n = 20 cells) or both H2A-GFP and a mutant H2B E76K mCherry fusion (n = 25 cells) with standard deviation envelopes. Inset demonstrates co-expression of H2A GFP and E76K mCherry. (E) Representative pre-bleach, bleach and post-bleach images of H2A GFP in WT MCF10A cells or in cells co-expressing mCherry tagged H2B-E76K. (F) 100nm particles injected into the nucleus had significantly increased mean square displacement (MSD) over time in MCF10A cells stably expressing exogenous H2B-E76K (red) compared to cells expressing WT H2B (blue). N=68 (WT) and N=67 (E76K) cells were analyzed. Error bars represent SEM.

    Article Snippet: MNase susceptibility assays were performed on nucleosomes made with WT, E76Q and E76K H2B mutants by mixing each nucleosome (0.4 pmol) with 9.6U of MNase (Worthington) and incubating for indicated amount of time.

    Techniques: Stable Transfection, Expressing, Agarose Gel Electrophoresis, Fluorescence, Mutagenesis, FRAP Assay, Standard Deviation, Injection

    The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and MNase digestion. The genomic DNA was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.

    Journal: PLoS ONE

    Article Title: Serum Starvation Induces DRAM Expression in Liver Cancer Cells via Histone Modifications within Its Promoter Locus

    doi: 10.1371/journal.pone.0050502

    Figure Lengend Snippet: The proximal region of the DRAM promoter is sensitive to nuclease digestion. (A) Schematic representation of the primer sets used for CHART-PCR. (B) R6 region of the DRAM promoter is more sensitive to DNase I and MNase digestion. The genomic DNA was isolated from Hep3B and HepG2 cells and analyzed by quantitative PCR. The data was shown as percentage of digested DNAs to undigested DNAs. (C) Serum deprivation increases the accessibility of R5, R6 and R7 regions to nuclease digestion in Hep3B and HepG2 cells.

    Article Snippet: Chromatin accessibility analysis Accessibility of DNA to DNase I digestion and MNase (Takara, Inc., Dalian, China) were analyzed using chromatin accessibility through CHART-PCR as described previously – .

    Techniques: Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction